WO2007081410A2 - Encoded microparticles - Google Patents

Abstract

An encoded microparticle, methods for using the same in bioassays, and a method of making the same are provided herein.

Description

ENCODED MICROPARTICLES

CROSS-REFEER]SfCE TO RELATED CASES

[0001] This patent application claims priority from co-pending US provisional application serial number 60/762,238 filed Jan. 25, 2006 and US provisional application serial number 60/716,694 filed Sept. 13, 2005, the subject matter of each being incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to the art of microstructures, and more particularly to encoded microparticles.

BACKGROUND OF THE INVENTION

[0003] Microparticles or nanoparticles are often referred to as structures whose characteristic dimensions are on the order of micrometers or less, such as those with volumes of 1 mm³ or less. Due to their unique properties arising from their small characteristic dimensions, microparticles have found distinguishable applications in laboratory research and many industrial fields. Encoded microparticles possess a means of identification and are an important subclass of the general field of microparticles. Because encoded particles carry information and can be physically tracked in space and time, they greatly extend the capabilities of non-encoded particles. A particularly important application for encoded microparticles is multiplexed bioassays, including those involving DNA and proteins. Other important fields for encoded microparticles include combinatorial chemistry, tagging, etc. Many biochemical and non-biochemical applications as will be discussed herein below. [0004] For many applications, one more desirable attributes include: a large number of identifiable codes (i.e. a high codespace), accurate and reliable identification of the encoded particles, material compatibility for a particular application, low cost manufacturing of the microparticles (on a per batch, per particle, and per code set basis), and flexibility in the detection systems.

[0005] Several approaches to produce encoded microparticles have been developed in the past, such as fragmented colored laminates, colored polystyrene beads, quantum dot loaded polymer beads, rare-earth doped glass microbarcodes, electroplated metal nano rods, diffraction grating based fiber particles, and pattern bars and disks, and other types of microparticles. These technologies however suffer from any of a number of limitations, such as, insufficient codespace, high cost, inadequate precision, poor performance in applications, problematic clumping incapability of large scale manufacture, and complicated preprocessing or assay procedures.

[0006] Therefore, what is desired is an encoded microparticle or a set of encoded microparticles carrying coded information, methods of making the same, methods for providing the codes for microparticles, methods for fabricating the microparticles, methods and systems for detecting microparticle, and methods and systems for using.

BRIEF DESCRIPTION OF DRAWINGS

[0007] While the appended claims set forth the features of the present invention with particularity, the invention, together with its objects and advantages, may be best understood from the following detailed description taken in conjunction with the accompanying drawings of which:

[0008] FIG. IA schematically illustrates an encoded microparticle of the invention; [0009] FIG. Ib is a side view cross-section of the microparticle in FIG. Ia; [0010] FIG. 2 schematically illustrates another example encoded microparticle of the invention;

[0011] FIG. 3a schematically illustrates another example encoded microparticle of the invention;

[0012] FIG. 3b schematically illustrates an another example encoded microparticle of the invention;

[0013] FIG. 4a and FIG. 4b schematically illustrates an exemplary microparticle whose coding structures are derived from a single material;

[0014] FIG. 4C schematically illustrates an another example encoded microparticle of the invention;

[0015] FIG. 4d is a cross-sectional view of another exemplary microparticle during an exemplary fabrication of the invention;

[0016] FIG. 5 is a flow chart showing the steps executed in an exemplary fabrication method of the invention;

[0017] FIG. 6a to FIG. 6m are cross-section views and top views of a microparticle in an exemplary fabrication process of the invention; [0018] FIG. 8 is a perspective view of an array of microparticles on a substrate during the fabrication;

[0019] FIG. 9a to FIG. 10 are SEM images of a plurality of microparticles during the fabrication of an exemplary fabrication method of the invention;

[0020] FIG. 11a and FIG. 1 Ib illustrate an exemplary etching method that can be used in the fabrication method of the invention;

[0021] FIG. 12a and FIG. 12b are images of a plurality of microparticles of the invention;

[0022] FIG. 13a to FIG. 13c schematically illustrate an exemplary wafer level fabrication method according to an exemplary fabrication method of the invention;

[0023] FIG. 14 presents a reflectance-mode inverted microscope image of 8 encoded microparticles of the present inventions;

[0024] FIG. 15 shows a diagram of an optical system used to image the encoded microparticles of the invention;

[0025] FIG. 16 presents a full field, single image taken at the same magnification as that in

FIG. 14;

[0026] FIG. 17 shows a high magnification image of encoded microparticles;

[0027] FIG. 18a shows a montage of 12 dense reflectance images of encoded microparticles;

[0028] FIG 18b shows a transmission' fluorescence microscope image of example microparticles of the invention;

[0029] FIG. 19A shows a full field reflectance image;

[0030] FIG. 19B shows the same image selection of FIG. 19A after the image processing to associate discrete segments into full microparticles;

[0031] FIG. 2OA shows a selection of a reflectance image;

[0032] FIG. 2OB shows the same image selection of FIG.2OA after the image processing to associate discrete segments into full microparticles;

[0033] FIG. 21 illustrates a processed image is shown on the right and pixel intensity profiles from 4 example microparticles are shown on the left;

[0034] FIG. 22 shows a schematic of a specially prepared surface that have features designed to immobilize and separate the encoded microparticles for imaging;

[0035] FIG. 23 and FIG. 24 show a flow-cell enabling the microparticles flowing in a fluid can be provided for detection by continuous imaging;

[0036] FIG. 25 illustrates another alternative microparticle of the invention; [0037] FIG 26 shows a diagram of a spatially optically encoded microparticle with a fluorescent outer layer;

[0038] FIGs. 27a to 27c show schematic diagrams of encoded microparticles of the present invention with surface indentations that form a spatial code;

[0039] FIG. 27d shows an example of encoded microparticles comprising indentations;

[0040] FIGs. 28a to 28c show the non-uniform aerial density measured normal to the particle surface for corresponding particles in FIGs. 27a to 27c;

[0041] FIG. 29a to FIG. 30c are top views of microparticles according to another example of the invention during another exemplary fabrication of the invention;

[0042] FIG. 31A to 31C show drawings of the 3 mask fields of the preferred embodiment of the microparticle structure and Fig 3 ID shows a drawing of a reticle plate;

[0043] FIG. 32 shows an alternate example of the general method of generating code using multiple print steps utilizes stamping;

[0044] FIG. 33 A to FIG. 33M illustrate the microfabrication process steps of the example encoded microparticle of FIG. IA;

[0045] FIG. 34a to FIG. 34m show the corresponding cross sectional views of the microparticle in FIG. 33a to FIG. 33m;

[0046] FIG. 35A to FIG. 35c show exemplary microparticles that can be produced using the method of the invention;

[0047] FIG. 36 shows four microscope images of actual encoded microparticles, just prior to release from the dies;

[0048] FIG. 37 shows charts of example data that is input into the stepper software to generate different codes on every die on a wafer;

[0049] FIG 38 shows drawings of an example scheme for producing an increased number of codes per die;

[0050] FIG. 39A shows a graphical representation of encoded microparticles that are formed according to the invented non-binary coding scheme;

[0051] FIGs. 39B and 39C show random codes with differnet numbers of gaps and gaps of varying location;

[0052] FIG.40 shows photographs a montage of 4 photographs of various forms of a large prototype set of microparticles;

[0053] FIG. 41 is a flow chart of an exemplary bioassay process; [0054] FIG. 42 shows a diagram of an exemplary example of the process by which whole wafers become mixtures of particle-probe conjugates that are ready to be reacted with samples to perform a bioassay;

[0055] FIG.43 shows a diagram of an optical system used to image encoded microparticles that utilizes two CCD cameras for the simultaneous acquisition of a reflectance and fluorescence image;

[0056] FIG. 44 and 45 show dense fluorescence microscope image of a multiplicity of encoded microparticles;

[0057] FIG. 46A and FIG. 46B show a reflectance and fluorescence image pair for the same set of microparticles of the invention;

[0058] FIG. 47A to FIG. 47F show dense fluorescence microscope images of encoded microparticles in a time sequence;

[0059] FIG. 48 shows real assay data from a 2-plex DNA hybridization assay;

[0060] FIG.49a illustrates an exemplary assay in which the microparticles of the invention can be used;

[0061] FIG. 49b illustrates another exemplary assay in which the microparticles of the invention can be used;

[0062] FIG. 50 illustrates another exemplary assay in which the microparticles of the invention can be used;

[0063] FIG. 51 is a schematic that includes images of particles but is not the result of an actual experiment of this invention; and

[0064] FIGs. 52A to 52C show flowcharts of examples of the code element patterning and etch steps.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0065] An encoded microparticle is provided carrying a code, and a set of encoded microparticles are provided with distinguishable codes, wherein the codes comply with a pre-determined coding scheme. Preferably, the microparticles in the examples below have a volume of 1 mm³ or less. The microparticle of the invention enables fast, precise and less complicated detection of the code. Methods for providing the codes on microparticles, methods for fabricating the microparticles, methods and systems for detecting the microparticle, and methods and systems for using the microparticles are also disclosed. [0066] In the following, the invention will be discussed with reference to specific examples. It will be appreciated by those skilled in art that the following discussion is for demonstration purposes, and should not be interpreted as a limitation. Instead, other variations without departing from the spirit of the invention are also applicable.

Overall Structure of the Microparticle

[0067] As an example, FIG. IA schematically illustrates an encoded microparticle of the invention. Microparticle 100 is a cuboid structure elongated along the Y direction in the Cartesian coordinate as shown in the figure. The cross-sections perpendicular to the length of the microparticle have substantially the same topological shape - which is square in this example.

[0068] The microparticle in this particular example has a set of segments (e.g. segment 102) and gaps (e.g. gap 104) intervening the segments. Specifically, segments with different lengths (the dimension along the length of the microparticle, e.g. along the Y direction) represent different coding elements; whereas gaps preferably have the same length for differentiating the segments during detection of the microparticles. The segments of the microparticle in this example are fully enclosed within the microparticle, for example within body 106. As an alternative feature, the segments can be arranged such that the geometric centers of the segments are aligned to the geometric central axis of the elongated microparticle. A particular sequence of segments and gaps represents a code. The codes are derived from a pre-determined coding scheme.

[0069] Segments of the microparticle can be any suitable form. In an example of the invention, each segment of the microparticle has a substantially square cross-section (i.e. the cross-section in the X-Z plane of a Cartesian coordinate as shown in FIG. IA) taken perpendicular to the length (/. e. along the F direction in the Cartesian coordinate in FIG. IA) of the microparticle. The segments may or may not be fabricated to have substantially square cross-section. Other shapes, such as rectangular, circular, and elliptical, jagged, curved or other shapes are also applicable. In particular, the code elements - i.e. segments and gaps, may also take any other suitable desired shape. For example, the segment (and/or the gaps) each may have a cross-section that is rectangular (e.g. with the aspect ratio of the rectangular being 2: 1 or higher, such as 4: 1 or higher, 10: 1 or higher, 20: 1 or higher, or even 100: 1 or higher, but preferably less than 500: 1). [0070] The microparticle example of FIG. IA has six major surfaces, namely surfaces of (X=±xo, Y_> Z), surfaces (X, Y, Z=±z₀), and surfaces (X, Y=±yo, Z), wherein xo, yo, and z₀ are respectively the width, length, and height of the microparticle. According to the invention, at least two of the above six surfaces X=±xo (or surfaces Z=-fczø), more preferably four of the above six major surfaces X=±xo, surfaces Z=±zo are substantially continuous, regardless of whether each surface has or does not have indentations. With this configuration, the microparticle exhibits substantially the same geometric appearance and specific properties to the detector - such as an optical imaging apparatus. In fact, the major surfaces can be made substantially flat. For example, even though roughness or varying profiles may be caused during fabrication, substantially flat major surfaces can still be obtained using standard surface machining techniques, such as over-deposit and etch back or chemical-mechanical-polishing (CMP) techniques, as well as proper control of patterning steps to create smooth vertical sidewall profiles.

[0071] The code elements , i.e. the segments and gaps, may take any desired dimensions. As an example of the invention, each coding structure has a characteristic dimension that is 5 um (microns) or less, such as 3 microns or less, and more preferably 1 micron or less, such as 0.8 or 0.5 microns or less. In particular, when gaps are kept substantially the same dimension while the segments vary in dimension, each gap preferably has a characteristic dimension that is 1.5 microns or less, such as 0.8 or 0.5 microns or less.

[0072] As one example, if forming the microparticles on a 12-inch silicon wafer with 0.13 line widths, the gap areas can be made to have 0.13 um minimum widths, with the less transparent segments having widths of from 0.13 um to much larger (depending upon the desired length of the particle and the encoding scheme and code space desired). Minimum gap widths, as well as minimum segment widths, of from 0.13 to 1.85 um (e.g. from 0.25 to 0.85 um) are possible depending upon the wafer fabrication used. Of course larger minimum gap and segment lengths (e.g. 1.85 to 5.0 um, or more) are also possible. Other sized wafers (4 inch, 6 inch, 8 inch etc.) can of course be used, as well as wafers other than silicon (e.g. glass), as well as other substrates other than silicon (larger glass panels, for example). [0073] Though the microparticle may have the same length in the X, Y and/or Z directions, preferably the encoded microparticle has a ratio of the length to width of from 2:1 to 50:1, e.g. from 4:1 to 20:1. In an example of the invention, the microparticle has a length (e.g. the dimension along the Y direction) of 70 microns or less, 50 microns or less, 30 microns or less, such as 20 microns or less, 16 microns or less, or even 10 microns or less. The width (e.g. the dimension along the X direction), as well as the height (the dimension along the Z direction), of the microparticle can be 15 microns or less, 10 microns or less, 8 microns or less, 4 microns or less, or even 1 microns or less, such as 0.13 micron. Widths as small as from 0.5 to 2 microns are also possible. Other than the shape as shown in FIG. IA and discussed above, the microparticle may take a form of rod, bar, disk or any other desired shapes. [0074] The coding structures and gaps of the microparticles can take any suitable form as long as the coding structures and gaps together represent detectable codes. As mentioned above, the cross-section of the microparticles, as taken perpendicular to the length of the particle, can be square, rectangular, circular, elliptical, or any desired shape such as jagged or curved shapes or other profiles. When the cross-section is rectangular, the rectangle preferably has an aspect ratio (the ratio of the length to the width or height) of 2: 1 or higher, such as 4: 1 or higher, 10:1 or higher, 20:1 or higher, or even 100:1 or higher, but preferably less than 500:1. The ratio of the width to height can be around 1 : 1 (square cross section), or have a ratio of from 1 :4 to 1:1 - preferably a ratio that allows the particle to rest on either the sides defining the width or height of the particle such that the code of the microparticle can be detected regardless of which of the elongated sides the particle rests.

[0075] To facilitate fast, cost-effective, reliable, and easy detection of the code represented by the coding structures and gaps, it is preferred that each coding structure is as omni-directional as possible to the detection means. That is - each coding structure exhibits substantially the same geometric appearance or detectable properties when observed from at least two directions, more preferably from four (or all, if not four-sided in cross section) directions perpendicular to the length of the microparticle. Accordingly, the coding structures preferably possess rotational symmetry along the length of the microparticle, such as 2-folded or 4-folded rotational symmetry.

[0076] A microparticle of the invention can have any suitable number of coding structures depending upon the shape or length of the particle, and the code space desired. Specifically, the total number of coding structures of a microparticle can be from 1 to 20, or more typically from 3 to 15, and more typically from 3 to 8.

[0077] The desired code can be incorporated in and represented by the microparticle in many ways. As an example, the coding elements of the pre-determined coding scheme can be represented by the segment(s) - e.g. segments of different lengths represent different coding elements of the coding scheme. Different spatial arrangements of the segments with the different (or the same) lengths and intervened by gaps represent different codes. In this code-incorporation method, the intervening gaps preferably have substantially the same dimension, especially the length in the direction to which the segments are aligned. As another example, the codes are incorporated in the microparticle by arranging gaps that vary in lengths; while the segments have substantially the same dimension and are disposed between adjacent gaps. In another example, the both segments and gaps vary in their dimensions so as to represent a code. In fact, the code can also be represented in many other alternative ways using the segments, gaps, and the combination thereof.

[0078] For representing a code derived from the predetermined coding scheme, the segments and gaps are arranged along the length (the Y direction) of the elongated microparticle (2D, or even 3D, arrangements however are also possible). Specifically, the segments and gaps are alternately aligned along the length with the each segment being separated (possibly fully separated and isolated) by adjacent gaps; and each gap is separated (possibly fully separated and isolated) by adjacent segments, which is better illustrated in a cross-sectional view in FIG. IB, which will be discussed in the following. [0079] In an example of the invention, any suitable number of segments can be used - e.g. from 2 to 20, or more typically from 3 to 15 segments (more typically from 3 to 8 segments) of less transparent material (as compared to the intervening gaps between the segments) are provided within the encoded microparticle. To form the code, it is possible that the segments of less transparent material are varying lengths. Alternatively, the segments of less transparent material could each have substantially the same length whereas the intermediate segments of more transparent material could have varying lengths. Of course, the segments of more transparent material and the intermediate segments of less transparent material could both have varying lengths in order to represent the code.

[0080] Referring to FIG. IB, the cross-section is taken in the Y-Z plane (or equivalently in the X-Y plane) of the particle in FIG. IA. Segments (e.g. segment 102) and gaps (e.g. gap 104) alternate along the length of the microparticle.

[0081] In order to enable detection of codes incorporated in microparticles, the segments and gaps in each microparticle can be composed of materials of different optical, electrical, magnetic, fluid dynamic, or other desired properties that are compatible with the desired detection methods. In one example the segments and gaps are directly spatially distinguishable under transmitted and/or reflected light in the visible spectrum. For example, when the code detection relies upon optical imaging, the distinguishable property (segments vs. gaps) can be a difference in transmissivity to the particular light used for imaging (which can be any desired electromagnetic radiation - e.g. visible and near-visible light, IR, and ultra-violet light. The segments can be made to be more light absorbing (or light reflecting) than the intervening spacing material (or vice versa). When the code detection relies upon the electrical property measurements, the property can be resistance and conductance. When the code detection involves magnetic methods, the properties can be inductance and electro-inductance. When the code detection involves fluid dynamic methods, the property can be viscosity to the specific fluid used in the code detection. Regardless of which specific property is relied upon, the segments and gaps are preferred to exhibit sufficient difference in the specific property such that the difference is detectable using the corresponding code detection method. In particular, when the code is to be detected by means of optical imaging, the segments and gaps are composed of materials exhibiting different transmissivity (in an optical transmittance mode) or reflectivity (in optical reflectance mode) to the specific light used in imaging the microparticles. For example, the segments of the microparticle of the less transparent material can block and/or reflect 30% or more, preferably 50% or more, or e.g. 80% or more, of the visible light or near visible light incident thereon.

[0082] Given the fact that transmissivity of electromagnetic radiation through an object varies with the thickness of the object, it is preferred that the segments that are capable of blocking and/or reflecting 30% or more, preferably 50% or more, or e.g. 80% or more (or even 90% or more), of the detection light; while the gaps between the coding structures are provided from materials and at dimensions that are capable of transmitting 50% or more, 70% or more, 80% or more, or even 90% or more of the detecting light. Alternatively, the segments and gaps are composed of different materials such that the ratio of the transmissivity difference is sufficient to detect the codeγ, e.g. is 5% or more, 10% or more, 20% or more, 50% or more, and 70% or more. The transmissivity is defined as the ratio of the light intensities of the passed light to the incident light.

[0083] The microstructure can be made of organic and/or inorganic materials or a hybrid of organic and inorganic material. Specifically, the gaps (which are preferably more transmissive to visible or near- visible light) and segments (which are preferably less transmissive to visible or near-visible light as compared to gaps) each can be composed organic or inorganic materials, or a hybrid organic-inorganic material. The segments can be composed of a metal (e.g. aluminum), an early transition metal (e.g. tungsten, chromium, titanium, tantalum or molybdenum), or a metalloid (e.g. silicon or germanium), or combinations (or nitrides, oxides and/or carbides) thereof. In particular, the segments can be composed of a ceramic compound, such as a compound that comprises an oxide of a metalloid or early transition metal, a nitride of a metalloid or early transition metal, or a carbide of a metalloid or early transition metal. Early transition metals are those from columns 3b (Sc, Y, Lu, Lr), 4b (Ti, Zr, Hf, Rf), 5b(V, Nb, Ta, Db), 6b (Cr, Mo, W, Sg) and 7b (Mn, Tc, Re, Bh) of the periodic table. However, preferred are early transition metals in columns 4b to 6b, in particular tungsten, titanium, zirconium, hafnium, niobium, tantalum, vanadium and chromium.

[0084] The gaps which are in this example more transparent, can comprise any suitable material that is more transparent than the segments. The spacing material can be a siloxane, siloxene or silsesquioxane material, among others, if a hybrid material is selected. The spacing material, if inorganic, can be a glass material. Thin film deposited silicon dioxide is a suitable material, with or without boron or phosphorous doping/alloying agents. Other inorganic glass materials are also suitable such as silicon nitride, silicon oxynitride, germanium oxide, germanium oxynitride, germanium-silicon-oxynitride, or various transition metal oxides for example. A spin on glass (SOG) could also be used. If an organic material is used for the gap material, a plastic (e.g. polystyrene or latex for example) could be used. [0085] Both the segments and the gaps can be deposited by any suitable methods such as CVD (chemical vapor deposition), PVD (physical vapor deposition), spin-on, sol gel, etc. If a CVD deposition method is used, the CVD could be LPCVD (low pressure chemical vapor deposition), PECVD (plasma enhanced chemical vapor deposition), APCVD (atmospheric pressure chemical vapor deposition), SACVD (sub atmospheric chemical vapor deposition), etc. If a PVD method is used, sputtering or reactive sputtering are possible depending upon the desired final material. Spin on material (SOG or hybrid organic-inorganic siloxane materials [0086] As a more specific example, the segments can be comprised of a any suitable silicon material such as CVD (chemical vapor deposition) deposited amorphous silicon. Polysilicon or single crystal silicon area also suitable as are a wide range of other materials as mentioned above. It is preferred, but not necessary, that the material selected for the segments has a high degree of deposition thickness control, low surface roughness, control of etching - both patterning and release (e.g. using a dry plasma etch for patterning and a wet or dry chemical etch for release), and CMOS process compatibility. The gap material can be CVD deposited silicon dioxide. The silicon dioxide may include doping/alloying materials such as phosphorous or boron. Temperature considerations may be taken into account in choosing a combination of more and less transparent materials for the segments and gaps. [0087] FIG. 2 schematically illustrates another example encoded microparticle of the invention. Particle 20 has a rectangular cross section and is of a substantially flat shape. For example the ratio of the height to the width of the microparticlecan be any desired ratio, e.g can be from 1:1.2 to 1:4 or more, etc.

[0088] FIG. 3a schematically illustrates another example encoded microparticle of the invention. Referring to FIG. 3 a, microparticle 116 is composed of a 1^st material 118 and 2^nd material 120. The two materials can be chemically different or have the same chemical composition but be different in another respect such as grain structure or thickness. The two materials are distinguishable with the desired detection scheme. In this example each material preferably fully traverses the cross section of the particle. An example process for creating this structure involves fabrication methods as described, including those from the IC/MEMS (Integrated Circuii/Micro-Electro-Mechanical Systems) fields, including variations on the patterning and etching methods disclosed herein below, and/or with high energy ion implantation.

[0089] FIG. 3b schematically illustrates an another example encoded microparticle of the invention. Referring to FIG. 3b, the microparticle is comprised of alternating segments of two different materials 40 and 42 that are surrounded by a third material 44, whereby the pattern of alternating segments forms a detectable code. Other example microparticles may contain more than two different materials in the interior of the particle. The particle may have any suitable cross sectional shape and in the example shown, is elongated.

[0090] In the examples as discussed above, the microparticle is composed of materials of selected distinguishable properties, such as distinguishable optical properties. In the example above, one material has a greater transparency or optical transmissivity than the other material, which difference is detectable under magnification. A specific example of the above is where one material is a light absorbing material, and the other material is a translucent or transparent material with greater light transmittance in the visible spectrum (or in another spectrum should a different detection system be used - e.g. UV, IR etc). In another example, one material is a light reflecting material whereas the other material is either light absorbing or light transmitting. A detectable difference where one material is more opaque and the other material is less opaque, or where one material is more reflective and the other material is less reflective, are within the scope of this example. As mentioned above, the alternating portions of opaque and transparent materials can be made of silicon and glass among other materials. Given the fact that transmissivity (and reflectivity) of almost all materials exhibit dependencies from the thickness of the material, the microparticle may be formed such that the coding structures {i.e. the structures representing coding elements of a code) are derived from a single material. FIG. 4a and FIG. 4b schematically illustrates an exemplary microparticle whose coding structures are derived from a single material, such as silicon.

[0091] Referring to FIG. 4a wherein a cross-sectional view of an exemplary microparticle is illustrated therein. Microparticle 206 comprises a set of coding structures (e.g. 210, 212, 208, and 214), the combination of which represents a code derived from a coding scheme. For incorporating the code, the coding structures have different profiles, such as widths while different structures with different widths are positioned at particular locations. For defining the coding structure and code detection afterwards, a set of gaps (e.g. gaps 212 and 214) with thicknesses less than the transmissivity threshold thickness (the threshold below which the material is visible to the particular light such as visible and near-visible light). Different from the example as shown in FIG. Ia, the coding structures are not fully separated or isolated. The code incorporated in the microparticle can be read based on the different transmissivity of the coding structure (e.g. 210 and 208) which, for example, are less transmissive than the adjacent gaps (e.g. 212 and 214) between the coding structures.

[0092] For facilitating the application of the microparticles, especially biological/biochemical/biomedical/biotechnology applications wherein the sample bio-molecules are to be attached to the surfaces of the microparticles, an immobilization layer may be desired to be coated on the surfaces of the microstructures.

[0093] FIG. 4b schematically illustrates a transmissive-mode image of the microparticle in FIG. 4a. Referring to FIG. 4b, dark regions 210, 208 respectively correspond to the coding structures 210 and 208 in FIG. 4a. White regions 212 and 214 respectively correspond to the coding structures 212 and 214 in FIG. 4a. Even though the material used in the more light transmitting and less light transmitting sections is the same, the transmittance profile can still allow for a detectable code. Such a microparticle in FIG.4A can be formed with a bottom layer of another material (e.g. silicon dioxide), and be coated with a second layer of another material (e.g. silicon dioxide) if desired. Such a microparticle can also be fully encased in a material (e.g. silicon dioxide) such that it has substantially the same rectangular parallel piped shape as the structure in FIG. Ia. FIG. 4C schematically illustrates an another example encoded microparticle of the invention. Referring to FIG. 4C, the microparticle comprises larger regions connected by narrower regions. The microparticle is surrounded by a material such that a code is detectable. [0094] The microparticle of FIG. 4A and FIG. 4C can be fabricated in many ways, one of which is schematically demonstrated in a cross-sectional view of the microparticle during the exemplary fabrication in FIG. 4D. Referring to FIG. 4D, substrate 216 composed of material (e.g. glass, quartz, or other suitable materials) that is transmissive to a particular light (e.g. visible or near-visible light) is provided. Detaching layer 217 is deposited on substrate 216. The detaching layer is provided for detaching the microparticles from the glass substrate afterward by etching or other suitable methods. The etching can be wet, dry, or plasma etching; and the detaching layer is thus desired to be composed of a material etchable with the selected etching method, as discussed hereinabove. As described for previous embodiments of the particle structures, the detaching layer may be omitted such that the particle is formed directly on the substrate and is subsequently released by a bulk etch of the substrate. [0095] A coding structure layer is deposited and patterned so as to form the coding structures, such as structures 218, 222, 220, 224. After forming the coding structures, surrounding layer 224 is deposited on the formed coding structures. Because the surrounding layer will be exposed to the target sample in the assay, it is desired that layer 224 is composed of a material that is resistant to chemical components in the assay solution wherein the microparticles are to be dispensed. Moreover, for holding the probe molecules, such as nucleic acids (e.g. DNA or RNA), proteins, antibodies, enzymes, drugs, receptors, or ligands, molecules on the surface of the layer, layer 224 is desired to be capable of immobilizing the probe molecules.

Fabrication process

[0096] The following exemplary fabrication processes will be discussed in reference to microparticles with segments and gaps, however it should be noted that the following methods are applicable to many other types of code elements.

[0097] The microstructure of the invention can be fabricated with a method that fall into the broad field of micro-machining, such as MEMS fabrication methods. MEMS use the techniques of the semiconductor industry to form microscale structures for a wide variety of applications. MEMS techniques typically, but not in all circumstances, include the deposition of thin films, etching using dry and / or wet methods, and lithography for pattern formation.

Because MEMS is an offshoot of the semiconductor industry, a vast worldwide manufacturing infrastructure is in place for cost-effective, high volume, precision production. Generally speaking, the more similar the full MEMS process is to existing integrated circuit processes, e.g. CMOS compatible, the more accessible this infrastructure is.

[0098] The microstracture of the invention can be fabricated in many ways, such as fabrication methods used for integrated circuits (e.g. interconnects) or MEMS. In the following, an exemplary fabrication method compatible with the MEMS fabrication for making a microparticle will be discussed with reference to FIG. 5 and FIG. 6A to FIG. 6M, wherein the microparticle comprises opaque segments that are composed of amorphous silicon, and visible light transmissive gaps that are comprised of silicon dioxide. It will be appreciated by those skilled in the art that the following fabrication discussion is for demonstration purposes only, and should not be interpreted as a limitation on the scope of the invention. In fact, many fabrication methods could be used without departing from the spirit of the invention.

[0099] Referring to FIG. 5, a silicon substrate is provided at step 122. Other substrates, such as glass wafers or glass panels could also be used (as will be discussed further herein below). Assuming a silicon substrate, on the substrate is deposited a silicon dioxide layer at step 124. The deposition can be performed with many suitable thin film deposition techniques, such as CVD, PVD, spin-on etc. as mentioned above. An amorphous silicon layer is then deposited on the SiO₂ layer at step 126 followed by deposition of a hard mask oxide layer at step 128. Though not needed, the use of a hard mask reduces photoresist coating problems cause by topology , particularly when the amorphous silicon layer is relatively thick (e.g. 1 um or more in thickness). The hard mask oxide layer is then patterned at step 130. With the patterned hard mask layer, the amorphous silicon layer is etched with a plasma etch so as to form the desired pattern at step 132. A top SiO₂ layer is then deposited on the patterned silicon layer at step 134 followed by patterning the silicon dioxide layer at step 136 to form separate (but still unreleased) microparticles. Then the microparticles are released from the silicon substrate at step 140 by a non-direction silicon etch that etches into the silicon substrate and causes the microparticles to be separated as individual particles. The flow chart in FIG. 9 as discussed above can be better demonstrated in cross-sectional views and top views of the microparticle at different steps. The cross-sectional and top views are schematically illustrated in FIG. 6a to FIG. 6m.

[00100] Referring to FIG. 6a, SiO₂ layer 146, silicon layer 148, and hard mask layer 150 are sequentially deposited on silicon substrate 142. Hard mask layer 150 is then patterned so as to form segment strips (e.g. 152 and 156) and gap strips (e.g. 154 and 158), as shown in FIG. 6b. The segment and gap strips formed from the patterning of the hard mask layer correspond to the segments and gaps of the target microparticle. The segment and gap strips are better illustrated in a top view of the microparticle in FIG. 6c. Referring to FIG. 6c, segment strips (e.g. 152 and 156) and gap strips (e.g. 154 and 158) are formed with layer 148 that is visible from the top.

[00101] The patterning of the layers can be done in many methods, one of which is photolithography that is widely used in standard fabrication for semiconductor integrated circuits and MEMS devices. The most common form of photolithography used in the MEMS industry is contact photolithography. A reticle (aka mask) is typically composed of a binary chrome pattern on a glass plate. The reticle is placed very near or in contact with a photoresist covered wafer (or other substrate). UV light is shone through the mask, exposing the photoresist. The wafer is then developed, removing the photoresist in the exposed regions (for positive-tone photoresist). The pattern on the reticle is thus transferred to the photoresist where it serves as a mask for a subsequent etching step.

[00102] Projection photolithography is another type of photolithography that is used exclusively in modern integrated circuit manufacturing. Instead of bringing the mask into physical contact, projection^photolithography uses a system of lenses to focus the mask pattern onto the wafer. The primary advantage of this system is the ability to shrink the mask pattern through the projection optics. A typical system has a five times reduction factor. In general, much smaller feature sizes can be printed with projection as compared to contact lithography. A projection photolithography system is also known as a step-and-repeat system (or stepper for short). The maximum pattern or field size on the mask is significantly smaller than the wafer diameter. The mask pattern is repeatedly exposed ("stepped") on the wafer forming an array of "dies". The stepping distance is the distance the wafer stage travels in X and Y between exposures and is usually equal to the die size. This typical scheme produces a non-overlapping array of identical dies, allowing for subsequent parallel processing of the dies on the wafer. [00103] The hard mask layer (150) is further patterned so as to form discrete areas, as shown in FIG. 6d and FIG. 6e. As shown in FIG. 6d, the hard mask layer 150 is patterned in the X and Y directions so as to form discrete hard mask areas (eg. areas 160, 162, 164, and 166 in FIG. 6e). These discrete hard mask areas will in turn be used to form discrete silicon areas in the layer below. [00104] In the example above, the patterning of the hard mask layer is performed in two separate lithography steps. In an alternative example, the reticle may comprise a pattern such that the patterning of the hard mask can be accomplished with a single lithography step. As a further alternative, the hard mask can be omitted and either a two step or single step lithography process used.

[00105] After patterning the top hard mask layer, silicon layer 148 is etched so as to form corresponding discrete silicon areas on the substrate, such as silicon segments 168 and 172, with areas there between for material of greater transparency (e.g. gap areas 170 and 172, as shown in FIG. 6f). The top view of the microparticle as shown in FIG. 6f is schematically illustrated in FIG. 6g. As seen in FIG. 6g, transmissive layer 146 is now exposed when viewed from the top, with segments 160, 162, 164, and 166 formed on transmissive layer 146. SEM images of the structures at this point in the fabrication process are shown in FIG. 9A and FIG. 9B. The structures have a very high degree of precision, e.g. vertical sidewalls and sharp corner. Of course more rounded structures are also in the scope of these methods. [00106] After patterning silicon layer 148, transmissive layer 168 is then deposited as shown in FIG. 6h. The more light transmissive layer 168 may or may not be composed of the same material as the more light transmissive layer 146. A top view of the microparticles in FIG. 6h is schematically illustrated in FIG. 6i. A perspective view of the particles on the substrate is shown in FIG. 8.

[00107] The microparticles are then separated from each other, while still attached to the underlying substrate, as shown in FIG. 6j. FIG. 6k schematically illustrates a top view of the microparticle in FIG. 6j, wherein each microparticle is separated from adjacent microparticles, but surrounded by the light transmissive layer {i.e. layer 168 in FIG. 6h). Finally, the separated microparticles are detached from the silicon substrate 142, as shown in a cross-sectional view in FIG. 61. A top view of the detached microparticles from the silicon substrate is illustrated in FIG. 6m. The detaching of the microparticles from the underlying substrate (the "release" step) can be performed with any suitable etchant - preferably a gas or liquid matched to etch in all directions and undercut the microparticles. An additional sacrificial layer can be provided on the substrate in place of etching into the substrate itself. The etching can be wet, dry, or plasma etching; and the detaching layer is thus desired to be composed of a material etchable with the selected etching method. In particular, the etchant can be a spontaneous vapor phase chemical etchant such as an interhalogen (e.g. BrF₃ or BrCl₃), a noble gas halide (e.g. XeF₂), or an acidic vapor such as HF. A liquid could also be used to release the microparticles, such as TMAH, KOH (or other hydroxides such as NaOH, CeOH, RbOH, NH₄OH, etc.), EDP (ethylene diamine pyrocatechol), amine gallate, -HF etches glass so that won't work HNA (Hydrofluoric acid + Nitric acid + Acetic acid), or any other suitable silicon etchant (when the substrate or layer to be removed in the release is silicon (amorphous silicon or polysilicon or single crystal silicon — or tungsten, tungsten nitride, molybdenum, titanium or other material that can be removed in a silicon etchant such as XeF₂). If the material to be removed is not silicon, then the etchant is naturally matched to the sacrificial material (e.g. downstream oxygen plasma for a photoresist or polyimide sacrificial layer, etc.).

[00108] The indentations are as a result of the particular fabrication method; and can remain in the final product, or can be removed by, for example, planarization — e.g. chemical-mechanical-polishing (CMP) techniques. In fact, the indentations in some situations can be beneficial for code detection and/or fluorescence quantitation using fluorescent methods because the binding of a fluorescently tagged material to the surface of the microbarcode is greater in the indentation areas (per unit length of the microbarcode), the so called indentation signal enhancement, fluorescence can be greater in the indentation areas and can be used to determine the code (with or without other transmissive or reflective techniques discussed herein below). The same indentation signal enhancement would be applicable with reporter systems other than fluorescence, e.g. radioactive reporters, etc.

[00109] Though a silicon wafer was mentioned as the substrate in the example given above, a glass substrate, such as a glass wafer or larger glass sheet or panel (e.g. like those used in the flat panel display industry) could be used. Glass (or silicon) wafers can be of any suitable size - e.g. 4 in., 6 in., 8 in. or 12 in. When a glass wafer is used, typically an additional sacrificial layer will first be deposited (for later removal during the release step). The sacrificial layer can be semiconductor material, such as silicon, an early transition metal, such as titanium, chromium, tungsten, molybdenum, etc. or a polymer, such as photoresist, as mentioned earlier herein.

SEMs

[00110] A scanning-electron-microscopy (SEM) image of a segment (e.g. segment 102) in FIG. Ia is presented in FIG. 9c. As can be seen in the figure, the cross-section of the segment is substantially square. The top of the segment has a width of 1.0 micron; and the bottom width of the segment has a width of 1.2 microns. The height of the segment is approximately 1 micron. Of course larger or smaller dimensions are possible. [00111] An SEM image of a multiplicity of microparticles fabricated with the exemplary fabrication method as discussed above is presented in FIG. 1OA. The SEM image clearly illustrates the opaque segment 172 surrounded by transmissive material of the microparticle. Also, the indentations mentioned previously are clearly visible. The sample in the SEM image of FIG. 1OA was prepared for characterization by cleaving a chip perpendicular to the long axis of the particles, followed by a timed silicon etch to provide higher contrast between the inner silicon and outer silicon dioxide, purely for imaging purposes.

Release

[00112] The microparticles of the invention can be fabricated at the wafer-level, and released either at the wafer level or die level. Specifically, a plurality of dies each comprising a set of microparticles can be formed on a wafer. The microparticles on each die may or may not be the same - that is the microparticles on each die may or may not have the same code. After forming the microparticles, the dies can be separated from the wafer; and the wafer(s) on the singulated dies can be then removed. An exemplary wafer-level fabrication method is demonstrated in FIG. 13A to FIG. 13C.

[00113] Referring to FIG. 13 A, a plurality of dies is formed on wafer 236. In this particular example, multiple microparticles are formed on each die. The number, such as 3, 221, or 967 on each die represents the code incorporated in the microparticles in the die. The microparticles can be formed with a method as discussed above with reference to FIG. 6A to FIG. 6M. After formation of the microparticles but prior to release, the wafer can be partially cut, preferably to a depth about half the wafer thickness. The wafer is then cleaned, for example with solvents and/or a strong acid (sulfuric, hydrogen peroxide combination). The clean is an important step as it prepares a fresh glass surface for later functionalization and biomolecule attachment. The clean can also be performed after the wafer is separated into individual dies, or on the particles once they have been released.

[00114] After the formation of the microparticles, the wafer is then broken into dies as shown in FIG. 13B, where each die preferably, but not necessarily, contains a single code. The dies are then placed in separate vessels such as test tubes or the wells of a well plate for release, shown in FIG. 13 C. The well plate can be a typical 96-well plate (or 24-well, 384-well, etc.), or any other suitable set of holding areas or containers. For example, dies containing the numerically represented codes: 3, 221, and 967, are placed in different tubes for release. By releasing, the microparticles are detached from the wafer; and the particles can fall into the solution in the releasing liquid when a wet etch is used. The microparticles over time settle to the bottom of the tube or well due to gravity (or the tubes can be centrifuged). In some applications, it may be desirable to release multiple dies comprising one or more codes into a single container.

[00115] FIG. 12A shows particles before release, and FIG. 12B shows the same particles (i.e. particles from the same die) after release. Both images are optical microscope images taken with a 10Ox air objective on a non-inverted inspection microscope. In Fig 12B the particles are dried on a silicon chip.

[00116] The releasing step can be performed in many ways, such as dry etch, wet etch, and downstream plasma etch. In an exemplary bulk wet etch, shown schematically in FIG. 1 IA tetramethyl ammonium hydroxide (TMAH) is used as the etching agent. TMAH can be heated to a temperature approximately from 70-80C. Other chemical etchants can also be used and may work equally well, such as interhalogen (e.g. BrF₃ and ClF₃) and noble gas halide (e.g. XeF₂), HF in spontaneous vapor phase etch, potassium hydroxide in a gas phase etch, KOH, and other suitable etchants. A screen having characteristic apertures (or filter membrane with pores) less than the smallest microparticle dimension can be placed on the top of each well or container, whether liquid or gas release is used, to keep the codes safely within each container and avoid contamination of microparticles into adjacent wells. During the etching, especially the gas phase etch or dry etch, a mesh can be attached to each tube, whether on one end of the tube, well or container, or multiple mesh covering on more than one side of a tube, well or container, such that gas etchant and etching products can flow freely through the mesh while the microparticles are stopped by the mesh. A mesh or other filter can help to drain the liquid release etchant as well, without releasing the microparticles. Another example of a release etch process is shown in FIG. 1 IB and involves the deposition or formation of a sacrificial layer, as has been previously described.

[00117] After pelleting the particles through centrifugation or lapse of time, the liquid (so called supernatant) is removed and the particles are washed several times in water or a solvent. "Washing" refers to the successive replacement of the supernatant with a new liquid, usually one involved in the next chemical processing step. After detaching the microparticles from the substrate (or wafer), the substrate can be removed from etchant - leaving the microparticles in tubes. The released microparticles can then be transferred to containers for use. [00118] The microparticles can be fabricated on the wafer level, as shown in FIG. 13a to FIG. 13c. Referring to FIG. 13a, wafer 236, which is a substrate as discussed above with reference to step 122 in FIG. 2, comprises a plurality of dies, such as dies 1 and 3. In an example of the invention, the wafer has 10 or more, 24 or more, 30 or more, or 50 or more dies. Each die comprises a number of microparticles of the invention, wherein the number can be 10000 or more, 20000 or more, or 50000 or more. The microparticles in the same die are preferably the same (though not required); and the microparticles in different dies are preferably different (again, not required) so as to represent different codes. In the instance when different dies comprise microparticles of different codes, the dies are preferably assigned with unique identification numbers, as shown in the figure so as to distinguish the dies and codes in dies.

Detection

[00119] FIG. 14 presents a reflectance-mode inverted microscope image of 8 encoded microparticles of the present inventions. All such black and white microscope images with a black background are taken on an inverted epi-fluorescence microscope with the released particles in the well of a well plate. The particles are dispensed into the well in a liquid and settle by gravity onto the bottom surface where they are imaged from below. Each particle in FIG. 14 has a different code. Segments of the less transparent material (e.g. opaque material in the visible spectrum), in this case amorphous silicon, reflect light and are the brighter regions in the image. The surrounding transparent material, in this case silicon dioxide, is not visible in the reflectance-mode images. The particles are 16um long by 2um wide and approximately square in cross section. The image is a combination of selections from 8 images, one for each code. The illumination light is at 436nm, and the objective used is a 6Ox magnification oil immersion lens.

[00120] FIG. 16 presents a full field, single image taken at the same magnification as that in FIG. 14. The image is a mixture of many different codes. All particles form a high density monolayer - that is, there is no particle aggregation or clumping. The characteristic of the monolayer formation is one of the key advantages of the microparticles of the invention. When the microparticles are overlapped, aggregated, or clumped, the microparticles can not be properly identified. As a consequence, microparticles that do not readily form monolayers as herein, are forced to be used at relatively low densities (the total microparticles per unit area on the imaging surface). Low density imaging translates to correspondingly low throughput for the number of particles measured per unit time. This low throughput can be a limitation in many applications

The tendency of the microparticles to form a monolayer is not trivial . Monolayer formation involves many factors, such as the surface charge state (or zeta potential) of the microparticles, the density of microparticles in a specific solution, the fluid in which microparticles are contained, and the surface onto which the microparticles are disposed. Accordingly, the microparticles of the invention are comprised of materials and are constructed in a form that favors the maintenance of a charged state sufficient to substantially overcome stiction forces; and thus microparticles are capable of undergoing Brownian motion which facilitates the formation of a reasonably dense monolayer of particles.

In biological applications, the microparticles are often used to carry biochemical probe molecules. For immobilizing such probe molecules, the microstructure preferably comprises a surface layer, such as a silicon dioxide layer, which can be chemically modified to attach to the probe molecules. In accordance with an example of the invention, the microparticles are constructed such that the microparticles are capable of forming a monolayer, for example, at the bottom of a well containing a liquid; and the monolayer comprises 500 or more particles per square millimeter, more preferably 1,000 or more, 2,000 or more, or 3,000 or more microparticles per square millimeter. In an alternative example, the microparticles can form a monolayer that such that the detectable particles occupy 30% or more, 50% or more, or 70% or more of the total image area (i.e. the image field of view). In connection with the example mechanism of self-assembled monolayer formation, it is preferred that the 2D diffusion coefficient of the microparticles of the invention is greater than IxIO^"12 cm²/s . For accommodating the monolayer of the microparticles, the container for holding the microparticles in detection preferably has a substantially flat bottom portion. [00121] FIG. 15 shows a diagram of an optical system used to image the encoded microparticles of the invention. The optical system 254 can be used to read the microparticle codes, including for bioassay applications. The system is an inverted epi-fluorescence microscope configuration. Other exemplary optical microscopy systems for the detection of the microparticles of the invention include but are not limited to confocal microscope systems, Total Internal Reflection Fluorescent (TIRF), etc. Well plate 257 contains many wells of which a single well 256 is imaged. The well plate sits on microscope stage 258. Microparticles that have been dispensed into well 256 in a liquid settle by gravity to the bottom surface. Light coming from light source 268 passes through excitation filter 266 which selects the illuminating wavelength. The illuminating light reflects off beam-splitter 262 and travels up through objective 260. Typically, only a fraction of well 256 bottom surface area is imaged. The imaged area is referred to as the "field" or "field area". Reflected or emitted light (know together as collection light) travels back down the objective and passes through the beam-splitter 262. Emission filter 270 selects for the collection wavelength. Finally the collected light is recorded with a detector 272, such as a CCD camera. This simplified version of the optical system is not meant to be complete. In practice, the actual microscope may have many more features, preferably including an automated stage and auto focus system for high throughput imaging. The excitation filter and emission filter can be mounted on computer controlled filter wheels and are automatically changed for the reflectance and fluorescence images. A computer controlled shutter controls the exposure times.

FIG. 43 shows a diagram of an optical system used to image encoded microparticles that utilizes two CCD cameras for the simultaneous acquisition of a reflectance and fluorescence image. The optical system is used for detection in bioassays. The system is an inverted epi-fluorescence microscope configuration. In the preferred embodiment, a wellplate 201 contains many wells of which a single well 203 is imaged. The wellplate 201 sits on the microscope stage 209. Particles that have been dispensed into the well 203 in a fluid settle by gravity to the bottom surface. Light coming from the light source 215 goes through the excitation filter 219 which selects the illuminating wavelength. The illuminating light reflects off the beam splitter 213 and travels up through the objective 211. Typically, only a fraction of the well 203 bottom surface area is imaged. The imaged area is referred to as the "field" or "field area". Reflected or emitted light (know together as the collection light) travels back down the objective and passes through the first beam splitter 213. The collection light then passes through the second beam splitter 217 which breaks it into the reflectance path and the fluorescence path. The emission filter 221 is located in the fluorescence path and selects for the appropriate fluorescence emission wavelength. The light in the fluorescence path is recorded with the fluorescence CCD camera 223. The light in the reflectance path is recorded with the reflectance CCD camera 225. This simplified version of the optical system is not meant to be complete. In practice, the actual microscope system may have more features, preferably including an automated stage and auto focus system for high throughput imaging. The excitation filter 219 and emission filter 221 may be mounted on computer controlled filter wheels to be automatically changed for multi-fluorophore experiments. A computer controlled shutter may be used to control the exposure times.

The system depicted in FIG.43 is an improvement over the standard one camera system that utilizes filter wheels (or filter cube wheels) to acquire reflectance and fluorescence images in succession. The invention is accomplished by splitting the outgoing beam path into two components with a beam splitter. One component is the reflectance path, which is captured with one CCD camera. The other component is the fluorescence path, which is filtered for the appropriate wavelength and captured with a second matched CCD camera. The beam splitter can be designed such that more light is directed into the fluorescence path such that the exposure times on the two cameras are approximately equal. The two camera system invention offers the advantage of increased throughput. Additionally, the invention offers the advantage of eliminating the positional shifts between reflectance and fluorescence images pairs that may be present in those of the one camera system. This simplifies the computer software based processing of image pairs because the particles are in the same physical locations in both images of the image pair. In a further embodiment, the optical system is used for detection in bioassays.

Fig 17 shows a high magnification image of encoded microparticles. The imaged particles consist of discrete segments of varying sizes. The smallest size segments 20 are 0.6um. End segments 22 form the end of a single particle. An exemplary example of the invention consists of encoded microparticles with spatial encoding features less than 1.5um in size.

FIG. 18a shows a montage of 12 dense reflectance images of encoded microparticles. Approximately 6,000 particles are in the images. The particles are a small fraction of the approximately 200,000 particles total in a well of a 384 wellplate. The total particles are approximately 10% of a set that contains 1035 codes (batches). The set was formed by combining approximately 2,000 particles from each of the 1035 batches where each batch contained approximately 2 million particles of a single code. These images are a subset of a larger image set from which data regarding identification accuracy is presented below.

FIG 18b shows a transmission fluorescence microscope image of example microparticles of the invention. Shown are here, in addition, small, elongated, encoded microparticles with an outer surface that is entirely glass. Shown are a multiplicity of non-spherical encoded particles with a silica (e.g. glass or silicon dioxide) outer surface and a length less than 70 um (e.g. less than 50um.). The length of the example particles in this particular example is 15 um.

In this image, the particles are in a solution that contains suspended fluorescent molecules. The fluorescent molecules, when excited by the microscope light source, provide illumination from above (i.e. behind with respect to the collection optics, see FIG. 15 for a diagram of the basic optical system) the particles. This image is similar to one that would be provided in transmission mode imaging configuration, and unlike the reflectance mode images of FIG. 16 to FIG. 18A, clearly shown the outer glass surface of the particles.

[00122] For successfully identifying the microparticles, e.g. reading the codes incorporated therein, the images of the microparticles may be processed. Such image processing can be performed with the aid of software programs. According to exemplary examples of software programs and algorithms, pairs of raw and processed image are presented in FIG. 19A and FIG. 19B and in FIG. 2OA and 2OB.

[00123] FIG. 19A shows a full field reflectance image; and FIG. 19B shows the same image selection of FIG. 19A after the image processing to associate discrete segments into full microparticles. The particles shown in the images are of a single code. Images of encoded microparticles of the present invention consist of discrete segments that appear white in the reflectance imaging. The gaps, which are between segments of individual microparticles consist of glass, are transparent, and therefore appear black in the reflectance image. The background of the images is also black. The segments are associated together into the particles by an algorithm. The algorithm finds the long axis of a long segment and searches along that axis for segments. Segments are accepted or rejected based on predefined parameters. The black lines in FIG. 19B correspond to particles for which segments have been associated together. In an exemplary example of the aforementioned algorithm, a computer program product that identifies the codes of encoded particles by associating discrete regions in an image into individual particles.

[00124] FIG. 2OA shows a selection of a reflectance image; and FIG. 20B shows the same image selection of FIG. 2OA after the image processing to associate discrete segments into full microparticles. The particles shown in the images are of a multiplicity of codes. The segments of the particles are numbered. The black lines in FIG. 2OB are drawn to illustrate the segments that have been grouped together into particles by the image processing software. [00125] Referring to FIG. 21, a processed image is shown on the right and pixel intensity profiles from 4 example microparticles are shown on the left. The pixel intensity profiles are further processed by a computer software program to determine the codes of the microparticles. By identifying the center locations of the gaps, as indicated by circles in the pixel intensity profile in the lower left, the codes of the microparticles can be identified. As mentioned previously, the center gap locations are not sensitive to variations in both the particle fabrication process or image processing, i.e. variations in the dimensions of the actual segments and gaps that make up the exemplary example structure of FIG. IA. This feature is highly advantageous as it provides robust and accurate code identification of the encoded microparticles.

[00126] Table 3 shows identification data for image sets that include those images shown in FIG. 18A.

TABLE 3

[00127] The microparticles included in Table 3 have a codespace of 30,069, wherein the codespace is defined as the total number of possible codes with the particular particle design, i.e. with the chosen coding scheme and coding scheme parameters. A pre-determined identification method assigns one of the 30,069 possible codes based on the analysis of the particle segment information. 1035 codes were randomly selected, manufactured, and mixed to form the collection. When analyzing the identification of the collection, if the software assigned code is one of the 1035, it is assumed to be correct. The number of "correctly" identified particles divided by the total is called the "ID%". This assumption underestimates the error rate (1 - ID%) by the probability that a random error falls within the 1035 present codes, or 1035 divided by 30,069 = about 3%. The assumption therefore ignores this 3% deviation and provides a close approximation to the true identification accuracy. [00128] FIG. 22 shows a schematic of a specially prepared surface that have features designed to immobilize and separate the encoded microparticles for imaging. The surface includes features, e.g. grooves and/or pits that trap the particles. Such surfaces could be useful in applications where the particles experience increased aggregation due to the nature of molecules coated on the surface or properties of the imaging medium. FIG. 22 shows an example of such a substrate 320 with grooves 322 designed to capture the particles. The substrate 320 is preferred to be glass, but may be other materials, for example other transparent materials. The grooves 322 shown in FIG. 22 have a V-shape but may take on any shape such as having a square or U-shaped bottom that accomplishes the task of capturing the particles. When particles are placed onto the surface, particles 324 fall into the grooves and are immobilized. In an exemplary example, encoded microparticles of the present invention, having an elongated and substantially square cross section, may be immobilized in grooves having a flat bottom.

[00129] In an alternate example, a flow-cell enabling the microparticles flowing in a fluid can be provided for detection by continuous imaging, as shown in FIG. 23 and FIG. 24. Referring to FIG. 23, reflectance and fluorescence image pairs are acquired with the optical system depicted in FIG. 6 while the well plate is replaced with flowcell 320. Encoded microparticles 322 flow in a carrier fluid. Flow may be driven by pressure (hydrodynamic) or electrical means (electro-phoretic or electro-osmotic). Further, microparticles may be aligned with electric or magnetic fields. The flow is from the left to the right as indicated by the arrow. The upper figure of FIG. 23 shows the flow cell at a given time and the lower figure of FIG. 23 shows the same flow cell at a subsequent time such that the particles have displaced a distance equal to approximately the length of the field of view. The optical system objective 330 is shown below the flow cell but may also be placed above the flow cell. In addition, the flow cell can be placed in other configurations with, for example, the flow being directed vertically. The objective 330 images the capture field area 328. The first field area 324 and the second field area 326 are shown as shaded regions. In the upper figure the first field area 324 overlaps with the capture field area 328 and therefore the first field area 324 is imaged. In the lower figure the second field area 326 overlaps with the capture field area 328 and therefore the second field area 326 is imaged. By appropriately matching the flow speed, flow cell size, and optical system, all particles passing through the flow cell can be imaged, thereby providing a system for high throughput detection. Another exemplary system for high throughput flow based detection of the encoded microparticles of the invention is a flow cytometer, the methods and applications thereof are well known in the art.

Other Structures

[00130] Another alternative microparticle of the invention is schematically illustrated in FIG. 25. Referring to FIG. 25, microparticle 274 comprises opaque segments, such as 276, and gaps, such as 278, which are transmissive to the visible or near-visible light. The opaque material can be composed entirely or partially of a magnetic material such as (but not limited to) nickel, cobalt, or iron. The magnetic material could be incorporated as a thin layer 280 sandwiched between another material that forms the majority of the opaque material. The magnetic material gives the particles magnetic properties such that they can be manipulated by magnetic fields. This can aid in particle handling or facilitate the separation of biomolecules. [00131] Fig 26 shows a diagram of a spatially optically encoded microparticle with a fluorescent outer layer 406. This invention has utility in the tagging of material goods whereby the fluorescent layer improves the ability to easily find and identify the particles against diverse backgrounds. In an exemplary example, the fluorescent outer layer 406 is grown using a modified version of the Stδber process [Van Blaadern, A.; Vrij, A.; Langmuir. 1992. Vol. 8, No. 12, 2921]. The fluorescent outer layer 406 makes the entire particle fluorescent and facilitates the finding of the particles during detection. The reading of the particle code can be accomplished by imaging the particle in reflectance or fluorescence mode. One may be preferred over the other depending on the application, medium in which or surface to which the particles are applied. Particles of a single code can be used or mixtures of particles of different codes can be used. The particles can be applied in a medium such as a lacquer, varnish, or ink. The particles may be used to tag paper or fibers. The particles may be used to tag objects made of metal, wood, plastic, glass or any other material.

[00132] In another example, the fluorescent layer may be comprised of fluorophores, or other luminescent materials. The fluorescent layer may interact with molecular species in an assay, for example with fluorescently labeled nucleic acids or protein samples via Fluorescence Resonant Energy Transfer processes. In yet another example, the microparticles may have a non-fluorescent layer, wherein incorporated in or on the layer are molecules, for example quenchers that interact with luminescent emitter molecules. [00133] FIGs. 27a to 27c show schematic diagrams of encoded microparticles of the present invention with surface indentations that form a spatial code. The microparticle may be fabricated by many methods including the aforementioned examples. FIG. 27a has surface indentations, aka divots, e.g. grooves, only on the of face of the structure. FIG. 27b has divots on two faces. In other examples, divots and other desirable surface features may be placed on one or more surfaces of the microparticle structures, so as to provide a spatial code. FIG. 27c shows another example of such a structure, whereby the overall shape of the microparticle is substantially cylindrical. In an example method of making the microparticle of FIG. 27c, optical fibers having a diameter less than lmm may be laser or tip scribed to form the indentations. The composition of the structures of FIGs. 27a to 27c may be selected from a wide variety of materials, with glass being a preferred example.

[00134] In exemplary examples of encoded microparticles comprising indentations, the surface of the particles have fluorescent, or otherwise emitting, molecules attached to or in the surface, as shown in FIG. 27d. The emitting molecules may be covalently attached to the surface, adsorbed to the surface, or otherwise bound to the surface. In an exemplary example, the emitting molecules are incorporated into a layer which is deposited onto the microparticle. A uniform surface coverage of emitting molecules, e.g. a constant number of fluorophores per unit area, results in a nonuniform aerial density. Aerial density is defined as an intensity per unit length or per unit area that is integrated through a depth of field in an optical image plane. In this example, the aerial density is measured as an signal intensity profile measured by a detector, for example a CCD camera or photomultipler tube. FIGs. 28a to 28c show the nonuniform aerial density measured normal (i.e. perpendicular) to the particle surface for corresponding particles in FIGs. 27a to 27c. The signal intensity profile has peaks corresponding to the location of the surface indentions of the particles, which thus provide a detectable and useful code. The surface features of the encoded microparticles of FIGs. 27a to 27c may be detected by methods other than the use of emitting molecules, including but not limited to the measurement of light scattering, e.g. darkfϊeld optical microscopy, etc.

Method for Producing Codes

[00135] The invented general method of generating the codes on microparticles consists of the use of multiple lithographic printing steps of a single code element per particle region. The multiple printing steps create multiple code elements per particle region. The code elements taken together form the code for the microparticle. In a preferred example, the printing steps are performed on many particles in parallel using a master pattern. A master pattern comprises an array of single code elements per particle region. A code element may represent more than one physical feature, such as holes, stripes, or gaps. The master pattern is printed multiple times such that a multiplicity of microparticles with complete codes is formed, wherein the multiplicity of microparticles comprises identical particles (e.g. all particles have the same code). Variations upon this theme, for example wherein the multiplicity of microparticles are not identical, are anticipated and will be described in detail below. Between multiple print steps, a component of the overall printing system changes to translate the code element within the particle region. In a most preferred example, this change is a movement of the substrate on which the particles are formed. In another preferred example, this change is the movement of the master pattern. In yet other examples this change is the movement of an optical element such as a mirror.

[00136] An exemplary example of the general method of generating code using multiple print steps involves photolithography as the printing mechanism, e.g. contact photolithography and projection photolithography. An exemplary example of projection photolithographic utilizes a step and repeat system (aka stepper). A reticle contains a code pattern that has a single code element per particle. Through multiple exposures of this code pattern at different lateral offsets, a multiplicity of code elements (per particle) is created. Combined, these code elements form a complete code. The lateral offsets define the code and are programmed into the stepper software. The offsets, and therefore the code, can be changed on a per die or per wafer basis. The codes printed on different dies on a wafer and/or different wafers in a lot are thus controlled by software and can be arbitrarily changed. This enables a powerful flexibility in the manufacture of large sets of codes. A single mask set, having one to a few masks, can be used to generate an arbitrary number of codes, numbering into the 10⁵ range and beyond. [00137] FIG. 29A to FIG. 29C shows an exemplary example of the invented method of producing the codes for microparticles. The microparticle regions 290 are areas that, upon completion of the fabrication process, will be discrete particles. FIG. 29A shows the status after the printing of the first code element 292 in each microparticle region 290, in this exemplary example the code elements are vertical stripes. FIG. 29B shows the status after the printing of a successive code element 294 in each mircoparticle region 290. FIG. 29C shows the status of the printing of three more code elements 296 in each mircoparticle region 290. The multiple printing steps thus provide codes on the microparticles. [00138] FIG. 30A to FIG. 3OC shows another example of the invented method of producing the codes for microparticles. The microparticle regions 300 are areas that, upon completion of the fabrication process, will be discrete particles. FIG. 30A shows the status after the printing of the first code element 302 in each microparticle region 300, in this exemplary example the code elements are circular. FIG. 3OB shows the status after the printing of a successive code element 304 in each mircoparticle region. FIG. 30C shows the status of the printing of three more code elements 306 in each mircoparticle region 300. The multiple printing steps thus provide codes on the microparticles.

[00139] Fig 3 IA to 31C show drawings of the 3 mask fields of the preferred embodiment of the microparticle structure andFig31D shows a drawing of a reticle plate. FIG. 31Ato 31C are small representative areas of the much larger full field (only 46 of approximately 2 million particles are shown). In these drawings, the regions that are gray have chrome on the actual reticle (so called "dark" in reticle terminology), and the regions that are white have no chrome (so called "clear"). Physically, the reticles are glass plates that usually measure 5" to 6.25" square and are about .09" thick. They are coated with a thin (a couple hundred run) layer of chrome. The chrome is patterned with a resist through a serial lithography process, usually using a laser or ebeam system. The reticle is then wet etched which selectively removes the chrome. The final reticle then consists of a glass plate with chrome on one side in the desired pattern.

[00140] The code pattern, shown in Fig 3 IA, has vertical stripes 110 that are clear. There is one vertical stripe per particle. Fig 3 IB shows the bar pattern, which consists of horizontal stripes 112 that are dark (or equivalently wider horizontal stripes that are clear). The outline pattern, shown in Fig 31C, consists of rectangles 114 that are dark. Clear streets 116 extend in the horizontal and vertical directions, separating the rectangles 114. The rectangles 116 will form the outer border of the particles. The horizontal stripes 112 define the width of the inner segments of opaque material. The vertical stripes 110 form the gaps in the segments. The gaps both form the code in the particle and separate two adjacent particles. Fig 16D shows a full reticle plate. The reticle field 118 is the center region of the reticle which contains the pattern to be exposed. Alternate examples of the patterns described are also envisioned, including combining the code and bar pattern into a single pattern that can used according to the described multi print method.

[00141] An exemplary example of the invented method for producing codes uses photolithography and positive-tone photoresit. Positive-tone means that the areas exposed to light are developed away. For a negative-tone resist, exposed regions are what remain after development. The photocurable epoxy SU-8 is an example of a negative-tone resist. In an alternate example using a negative-tone resist such as SU-8, the regions that are to be segments are exposed to light instead of the regions that are to be gaps.

[00142] FIGs. 52A to 52C show flowcharts of examples of the code element patterning and etch steps. FIGs. 52A shows the case where a hard mask is not used. This process is simpler but may produce segments with rounded corners because of the proximity effect of the photoresist exposures. At the corners of the segments, the photoresist gets some residual exposure from both the vertical stripes of the code pattern and the horizontal stripes of the bar pattern. The resulting rounding of the corners, though within the scope of the invention, is less desirable because it produces final particles that look different from the side vs. the top and bottom surfaces. The extent to which the rounding occurs depends on the specifics of the photolithography process including the pattern on the reticles, wavelength of the light source, and photoresist. FIG. 52B shows an exemplary example of the multi print method based patterning process and is described in detail in the below FIGs. 33A to 33M and FIGs. 34A to 34M. FIG 52C shows another example of the particle fabrication process where instead of transferring the bar pattern to the hard mask, the bar pattern photoresist is used as the mask in conjunction with the hard mask oxide. This example method eliminates a few steps but may not be appropriate depending upon the specifics of the poly etch chemistry. [00143] An alternate example of the general method of generating code using multiple print steps utilizes stamping (aka imprint lithography) as the printing mechanism, and is schematically depicted in FIG. 32. FIG. 32 schematically shows a small region of an example master pattern for stamp printing according to the invented multi-print-steps-to-build-the code-up method, e.g. 1) stamping or pressing a stamper apparatus into the particle containing substrate, followed by 2) moving either the stamper apparatus or the substrate, and 3) stamping at least one more time in a nearby location, such that a complete code on the microparticles is formed. The substrate on which the microparticles can be formed using imprint lithography maybe a wafer, such as a 100mm,150mm,200mm, or 300mm silicon wafer, or a panel, such as a 5" or larger glass or quartz panel, or rolled sheets (including but not limited to polymeric sheets).

[00144] Fig 33 A to 33M and 34A to 34M illustrate the microfabrication process steps of the example encoded microparticle of FIG. IA. These steps define the inner opaque segments (which contain the code). The steps are shown in more detail than in Fig 6a to FIG. 6m and include the photoresist exposure and development. Fig 33a to FIG. 33m show top down drawings and Fig 34a to FIG. 34m show the corresponding cross sectional views. The cross-section line 50 is shown in Fig 33A to FIG. 33M. In Fig 33 A, the top surface is the hard mask oxide 58. In Fig 34A, the film stack on the starting substrate 52 consists of the bottom oxide 54, poly 56, and hard mask oxide 58. In Fig 33B the wafer has been coated with unexposed photoresist 120. The unexposed photoresist 120 is shown as the top layer in Fig 34B. In Fig 33C and 34C, the unexposed photoresist 120 has been exposed with the code pattern a single time, forming exposed photoresist 122 regions. In Fig 33D and 34D, the code pattern has been exposed multiple times with lateral offsets applied between the exposures. In the preferred embodiment, the code pattern is exposed twice in directly adjacent regions to form double width stripes 124. Single width stripes 126 are the "gaps" that form the code. The double width stripes 124 are located in between the particles and separate the particles. To clarify, the lateral offsets are achieved by moving the stage on which the wafer sits. The lateral offsets are programmed into the stepper software. The lateral offsets define the code of the microparticles on that die. The lateral offsets (and thus code) can be different for every die on a wafer. Each wafer in a lot of wafers can have a different set of codes. In this way, very large code sets can be realized.

[00145] Fig 33E and 34E show the wafer after development of the photoresist. The exposed photoresist 122 from Fig 33D and 34D is removed revealing the underlying hard mask oxide 58. Fig 33F and 34F show the wafer after the oxide etch. The oxide etch removes the hard mask oxide 58 in the exposed regions revealing the underlying poly 56. Fig 33G and 34G show the wafer after the unexposed photoresist 120 of Fig 33F and 34F is removed. The hard mask oxide 58 is present in the regions that will become the segments. The poly 56 is exposed in the regions that will become the gaps in the opaque material. Fig 33H and 34H show the wafer after it is again coated with unexposed photoresist 120.

[00146] Fig 331 and 341 show the wafer after the exposure of the bar pattern. This is just a single exposure and is the same on all dies. This exposure is preferably aligned to the pattern already on the wafer. After exposure, the unexposed photoresist 120 pattern consists of horizontal stripes which define the segment width. The exposed photoresist 122 pattern consists of horizontal stripes which define the horizontal separations between the segments. Fig 33 J and 34 J show the wafer after the development of the photoresist. The exposed photoresist 122 from Fig 331 and 341 is removed revealing the underlying hard mask oxide 58 and poly 56. Fig 33K and 34K show the wafer after the oxide etch of the hard mask oxide. Only the poly 56 is present in the exposed photoresist region of Fig 331. Fig 33L and 34L show the wafer after the unexposed photoresist 120 is removed. At this point in the process, the top surface of the wafer is poly 56 with hard mask oxide 58 covering the poly 56 in the regions which are to become the segments of opaque material. Finally, Fig 33M and 34M show the wafer after the poly etch. The poly etch removes the poly 56 of Fig 33L and 34L, revealing the underlying bottom oxide 54. The hard mask oxide 58 is still present on the top surface of the poly 56 in the segment pattern.

[00147] In addition to the microparticle as illustrated in FIG. IA, the methods above can be used to produce the codes for other encoded microparticle designs including currently known particle designs as well as other alternative designs. The method above can be used to produce the codes for the encoded microparticles, for example, in FIGs. 35A to 35C. [00148] Referring to FIG. 35A, a bar-shaped microparticle with code elements consisting of holes such as holes 178 and 180 that are surrounded by frame material 182. The number and the arrangement of the holes forms a code derived from a predetermined coding scheme. [00149] FIG. 35B shows another bar-shaped particle with the code elements comprising notches, such as notch 196. The adjacent notches define a set of protruding structures with different widths. The total number of protruding structures and the arrangement of the protruding structures with different widths represent a code derived from a coding scheme. FIG. 35C shows a square plate shaped particle with the code elements consisting of holes, such as holes 200 and 202 that are separated by gap 202. The plate particle also includes an indentation 198 in one corner to break the symmetry of the particle and thus allow for more codes. Further shapes and code element architectures can also be made with the aforementioned method of producing codes.

[00150] FIG. 36 shows four microscope images of actual encoded microparticles, just prior to release from the dies. These particles are produced according to the invented technique of producing codes with multiple print steps and according to designs described above. [00151] FIG. 37 shows charts of example data that is input into the stepper software to generate different codes on every die on a wafer. The charts show which dies get printed in 9 different passes and with what offsets. The data shown in FIG 37 is an example of one system for organizing the multi print method using a stepper for providing a multiplicity of codes on a multiplicity of dies on a wafer. In this example, each die is exposed at most one time during a single pass. A wafer map of which dies are to receive exposures during the stepper exposure passes in this example is shown in the column on the left. "1" designates exposure. "0" designates no exposure. The middle column shows a wafer shot map of the exposure offsets, designated with offset letters "A","B","C", and "D". The right column shows a lookup chart of 1) the exposure location relative to the end of the particle, 2) the offset letter, and 3) the exposure locations programmed relative to a stepper reference point. The rows correspond to the different passes, 9 in this example.

[00152] Another example of a system for organizing the multi print method using a stepper is to exposure all of the code elements within a single die before moving on to the next die. Of course, a number of offsets other than four could be used. Though this and other examples of the general method of producing codes on microparticles has been described with respect to using a projection photolithography and a stepper, contact lithography and other patterning methods may also be used.

[00153] FIG 38 shows drawings of an example scheme for producing an increased number of codes per die. In this scheme, within a die there are fixed and variable code element locations. Dies are divided into sub regions where each sub region has a different pattern of fixed code elements. For each die, a different pattern of variable code elements is exposed. The fixed and variable code elements together make up the entire code. A single wafer thus contains a total number of codes equal to the product of the number of dies per wafer and sub regions per die. An individual die, containing sub regions of different codes, could be physically separated into smaller sub-dies and the different codes released into different tubes. An alternative is to keep the dies intact and release the whole die into a single tube .This would create a mixture of codes from the different sub regions. This approach may be particularly useful for combinatorial synthesis applications.

[00154] The invented method of producing codes, for example the use of a photolithographic step and repeat system to form a complete code through multiple exposure steps of a single reticle field, may be used to apply unique codes to many types of components, e.g. MEMS and IC devices.

Coding Scheme

[00155] The microparticles as discussed above have incorporated therein codes derived from any desired coding scheme, such as binary or non-binary coding. [00156] By way of example, FIG. 39A shows a graphical representation of encoded microparticles that are formed according to the invented non-binary coding scheme. Referring to FIG. 39 A, the coding scheme parameters are Z (the length of the particle), w (the width of the gap between segments), and d (the delta in the position of the gap center of the gap). FIG. 39A shows 4 particles with different codes such that only one of the gaps is varied in location. The gap is varied by amount equal to d, showing "adjacent" codes (e.g. codes that are similar and therefore more likely to be mis-identified for one another. FIGs. 39B and 39C show random codes with different numbers of gaps and gaps of varying location. Table 1 presents the total number of codes (codespace) for a variety of different parameter combinations. The number of codes is calculated from a computer software program that implements the invented non-binary coding scheme. Code degeneracy is taken into account in the algorithm (e.g. a pair of codes, such that when one is reversed, the codes are equivalent and the two codes are considered a single code). The parameters in Table 1 and Table 2 are specified in lOOnm units. The parameter combination Z=I 52, H>=8, tf=4 which gives 30,069 is shown in FIGs. 39A to 39C. Table 2 presents the total number of codes that can be represented by the microparticles by different Z. In an exemplary example, the discretization distance w is equal to or smaller than the characteristic segment size. As shown in Table 2, very large codespaces are available, and practically achievable with the aforementioned methods. The parameter combination Z=I 52, w=5, d=4 has a codespace of approximately 2 million.

TABLE 1

TABLE 2

[00157] In a exemplary example, the coding scheme utilizes code elements placed at locations spanned by interval lengths smaller than the code element size itself. This deviates from the standard binary coding where the code consists of the absence or presence of a feature at discrete, evenly spaced locations. In the preferred embodiment of this coding scheme, naturally applicable to the above structure manufactured using the multiple print technique, the code element is the gap in the segmented inner opaque material. The gap size is chosen to be one that is reliably defined by the stepper and photolithography process and also resolvable by the microscope (working at the desired magnification). The gap size, interval length, and particle length determine the codespace (number of codes possible). The determination of a codespace involves tradeoffs between particle density on the wafer, identification accuracy, optical detection system complexity, and particle number per microscope image. Codespaces of over a million can be produced and accurately identified using practical parameter combinations.

[00158] In the example of a standard binary coding scheme, the particle would be divided into units of equal length. Each unit could then be black or white, 0 or 1. Because the particle is symmetric, there are two codes that are the same when one is reversed (so called "degenerate" codes). When counting the codes, one from each of the pair of degenerate codes is preferably discarded. Without the degeneracy, there would be 2^N possible codes, where N is the number of bits (units). With the degeneracy, there are about half that number. Exactly, the number of possible codes with the standard binary format is [2^N + 2^{floor[(N+1)/21}]/2. In the example of the high contrast encoded microparticle structures of the present invention, previously shown in FIG. 14, FIG. 17, etc., within the full set of codes, there may be individual codes that have long runs of black or white regions. The black of the particles is indistinguishable from the black of the background, giving the particles extremely high contrast. However, codes having long runs of black are less desirable (though certainly within the scope of the invention) because it is more difficult to associate the white regions into the separate particles. For example, a more difficult code would be 1000...0001 (single white bits at both ends). It should be noted that, particularly for the structures and methods of making mentioned earlier herein, any suitable coding scheme can be used, as many other coding schemes are possible beyond that discussed in the example above.

[00159] The non binary coding scheme mentioned above has many advantages in the fabrication and detection of microparticles, including providing for high codespaces and robust code identification. In the example of the coding scheme, the reliability of the microparticle fabrication process is improved by permitting optimization of patterning and etch conditions for features, of a single size, e.g. gaps in the segments having a single width. [00160] In the exemplary examples of encoded microparticles and methods of determining codes therein, e.g. as shown in FIG. 21, the code is determined by the center location of the gaps and not the lengths of segments. Therefore, if the dimensions change, either because of variation in the manufacture or variation in the imaging conditions or variation in the image processing algorithm used, the center position of the gaps does not change, rendering the code ID is robust. This scheme exploits the fact that in an optical imaging system the position of features, in this case the gaps, can be located to a resolution much smaller than the minimum resolvable dimension of the features themselves. For example, if the gap width may be 1.5um or less, and located to a distance smaller than 1.Oum, more preferably smaller than 0.5um. [00161] In general, a high codespace is desirable. In the field of genomics, having a codespace in the tens of thousands is especially important because it enables full genomes of complex organisms, such as the human genome, to be placed on a single particle set. The top portion of Table 1 shows the effect of varying the delta parameter, d, on the codespace. Shrinking d gives many more codes but places increased demand on the optical system. The need to resolve a smaller d means that a more expensive objective would typically be used. Practically, the lower limit of the gap interval distance is set by the resolution by the optical system (manifested as the pixel size of the digital image captured using a CCD camera). Using a 60x objective and 6.2mm 1024x1024 CCD chip, an interval distance of d=0.4um equals approximately 4 pixels. If the interval distance is reduced to 0.3um (3 pixels), there are 105,154 codes. The codespace can be extended into the millions for longer particle lengths, L, and/or smaller gap widths, w.

[00162] The lower portion of Table 1 shows the effect of varying the length of the particle at fixed w and d. The length L is inversely proportional to the density of particles on the die (number of particles per unit area).The length also affects the number of particles in an image and thus throughput (particles detected per second). Tradeoffs exist between codespace, density, identification, and throughput. Optimization of the coding scheme parameters will determine the selected coding scheme for a particular application.

Large Particle Sets

[00163] FIG. 40 shows photographs a montage of 4 photographs of various forms of a large prototype set of microparticles. The set contains over 1,000 codes and approximately 2 million particles of each code. The upper left photograph shows 40 wafers during the fabrication process. Each wafer has 32 dies with each die comprising approximately 2 million particles of a single code. As a further example, dies on a wafer may contain many more particles per wafer, e.g. 5 million or more. Also, wafers (or other substrates, such as glass panels), may contain 100 or more dies, or alternately 200 or more, or 1000 or more dies. The wafer taken in whole may have 100 or more codes of encoded microparticle, or alternately 200 or more, or 1000 or more codes, or 5,000 or more codes. In an exemplary example of a large set of encoded microparticles, substantially all dies used to produce the large set, e.g. microparticles released from dies, comprise different codes. In another example, all dies on a wafer or substrate, may have the same code. The size of dies may be selected so as to optimize the balance between the number of particles per code and the number of codes in the large set of a large set. The number of particles per die and dies per wafer may be changed in software, for example by utilizing the invented method of producing codes, and optimized on a per manufacturing lot or per product basis for different applications, without necessitating the high capital costs of fixed tooling, e.g. large and expensive sets of photomasks. [00164] In the upper right photograph of FIG. 40, the wafer fabrication has been completed and the particles released from the silicon substrate into test tubes. The test tubes are shown in the photograph in placed in containers that each hold 64 test tubes. The photograph in the lower left corner shows a single test tube which contains a small portion (approximately a few thousand particles) of each of 1035 test tubes of particles from the large set. The lower right image is a microscope image of a sample of the single test tube. This image shows members of 1035 codes mixed together.

Assays

[00165] The encoded microparticles, systems, and methods of the invention have a wide range of applications in the fields of biology, chemistry, and medicine, as well as in security and commercial fields involving the tagging of monetary bills, identification cards and passports, commercial products , and the like. In one example, the microparticles can be used in for molecular detection, such for as analyzing DNA, RNA, and proteins. In other examples, combinatorial chemistry or drug screening assays are performed as known in the art. [00166] Referring to the flowchart shown in FIG. 41, microparticles are contained in separate tubes (or wells of well plates). Each tube contains a large number (e.g. a million or higher) of microparticles of a single code, at step 410. Biomolecules, such as DNA or RNA are immobilized on the surface of the particles and referred to as "probes" at step 412. Each ^' species of probe is immobilized onto a different code and a lookup table is generated for future reference. Each species of probe also has one or more corresponding species of "targets" for which the binding between the two is specific. The probe/target terminology is usually used in reference to DNA and RNA complements but in this context refers to all biomolecules, including antibodies. Many probes are immobilized on a single particle, typically with a density on the order 10⁴/um² or higher. The singular use of "a probe" often refers to a plurality of probe molecules; and "a code" often refers to a plurality of particles of a certain code, as with other terms used herein.

[00167] The mating of the encoded particles and biomolecules produces a "pooled probe set" through step 414. The pooled probe set is a mixture of encoded particles where each code has a particular probe attached to the particle surface. The pooled probe set can then be used to determine the amount of individual targets present in a mixture of targets. The mixture of targets is referred to as the sample and is typically derived from a biological specimen. The sample is then labeled, typically with a fluorophore at step 416. When the sample is mixed with the pooled probe set, the probes and targets find each other in solution and bind together. With nucleic acids, this reaction, step 418, is called hybridization and is very selective. After the reaction, the particles are imaged to read the codes and quantify the fluorescence at step 420. Referring to the code-probe lookup table, the amounts of the different target species in the mixed sample can now be measured and as a the assay result determined at step 422. [00168] The samples reacted with the microparticles may be a purified biological extract or a non-purified sample, including but not limited to whole blood, serum, cell lysates, swabs, or tissue extracts. The samples reacted with the microparticles may be produced by culturing, cloning, dissection, or microdissection. Cells may serve as either the sample or probe in a bioassay utilizing the microparticles and other aforementioned inventions. [00169] FIG. 44 and 45 show, dense fluorescence microscope image of a multiplicity of encoded microparticles. The microparticles shown in the images have oligo probe molecules attached to their surfaces and have been hybridized to pre-labeled fluorescent oligo targets, where the base pair sequence of the targets is complementary to the sequence of the probes. [00170] FIG. 42 shows a diagram of an exemplary example of the process by which whole wafers become mixtures of particle-probe conjugates that are ready to be reacted with samples to perform a bioassay (so called "Hybridization-Ready CodeArrays"). After completion of the wafer fabrication steps, the wafers has many dies where each die contains many particles of a single code. As has been previously described, alternative schemes may be used where dies are produced with the same code or dies are subdivided and contain multiple codes. The wafer is diced (usually by wafer saw) into the separate dies, then each die is placed into separate wells of a wellplate. Alternatively, test tubes can be used instead of wells. A release step is performed e.g. using a chemical etchant such as TMAH) that removes the particles from the surface of the die. The die is then removed from the well, leaving the free particles. After release, the conjugation of the biomolecule probes is performed resulting in each well containing a single type of particle probe conjugate (with particles of a single code and those particles having a single species of biomolecule on the surface). After conjugation, all of the particles are mixed together to form a "pooled master mix". The pooled master mix is divided into aliquots such that sufficient representation from all species of particle-probe conjugates is present. These aliquots are then ready to be reacted with a sample to perform a bioassay. [00171] It is noted that multiple different samples may be identified in a single bioassay as discussed above. Before the detection and after the hybridization, the microparticles can be placed into wells of a well plate or other container for detection. In one detection example, the microparticles settle by gravity onto the bottom surface of the well plate. The microparticles in the well can be subjected to centrifugation, sonication, or other physical or chemical processes (multiple washing steps, etc.) to assist in preparing the particles for detection. In another example, the microparticles can be placed onto a glass slide or other specially prepared substrate for detection. In yet other examples, the particles are present in a flow stream during detection, or present in a suspended solution.

[00172] Term conjugation is used to refer to the process by which substantially each microparticle has one or more probe molecules attached to it's surface. Methods of conjugation are well known in the art, for example in Bioconjugate Techniques, First Edition, Greg T. Hermanson, Academic Press, 1996: Part I (Review of the major chemical groups that can be used in modification or crosslinking reactions), Part II (A detailed overview of the major modification and conjugation chemicals in common use today), and Part III ( Discussion on how to prepare unique conjugates and labeled molecules for use in applications). [00173] The molecular probes attached to the surface of the particles typically have known attributes or properties. In an example, the molecular probes can be derived from biological specimens or samples and used in the screening, including but not limited to genetic sequencing, of large populations where typically, the derivatives from one member of the population is applied to a single code, typically a multiplicity of particles of a single code. Preferably, microparticles having the same code have attached substantially the same probe molecules; whereas microparticles having different codes likewise have different probe molecules.

[00174] One of the most powerful features of a multiplexed assay using solution arrays of encoded particles as the platform instead of planar microarrays is the flexibility to add functionality to the assay by simply adding new particles. With standard microarrays, once the arrays are printed or synthesized, the array typically cannot be changed. If the researcher wants to change the probes for genes on the array or add probes for new genes, typically entirely new arrays would then be produced. With pooled probe sets of particles, new probe and particle conjugates (probes for short) can easily be added to the existing pooled probe set. In practice the new probes could he different probes for an already represented gene, probes for alternative splicing variants of genes, or tiling probes for genes.

[00175] FIG. 46A and FIG. 46B show a reflectance and fluorescence image pair for the same set of microparticles of the invention. The images were taken in succession by about 1 second apart. FIG 46A, the reflectance image was taken with blue light illumination and collection (excitation filter = 436/1 Onm, emission filter = 457/50, i.e. overlapping filters). This image is used to determine the code of each particle. FIG. 46B, the fluorescence image, was taken with green illumination and red collection (excitation filter = 555/28nm, emission filter = 617/73, i.e. filters for Cy3). FIG.46C the image pair of FIG.46A and FIG.46B overlaid on top of one another in a single image.

[00176] FIG. 47A to FIG. 47F show dense fluorescence microscope images of encoded microparticles in a time sequence. The images have been processed for edge detection. The images were acquired approximately 1 second apart and are frames of the time sequence. The individual particles that comprise the images move a measurable amount between the frames due to molecular collisions (aka Brownian motion). This Brownian motion facilitates the assembly of the particles into a dense 2-dimensional monolayer. The particles shown in the images are examples of biochemically active encoded microparticles. The particles have oligonucleotide probes attached to the surface and have been hybridized (i.e. reacted in solution) with complementary oligonucleotide targets.

A bioassay process using the microparticle

[00177] The microparticles of the invention can be used as major functional members of biochemical (or chemical) analysis systems, including but not limited to solution based arrays, biochips, DNA microarrays, protein microarrays, lab-on-a-chip systems, lateral flow devices (immunochromatographic test strips). Applications include but are not limited to gDNA and protein sequencing, gene expression profiling, genotyping, polymorphism analysis, comparative genomic hybridization (CGH), chromatin immunoprecipitation (CHiP), methylation detection, as well as discovering disease mechanisms, studying gene function, investigating biological pathways, and a variety of other biochemical and biomolecular related applications such as inspection and analyses of proteins, peptides, polypeptide, and related biochemical applications. Assay architectures may include those well known in the art, including but not limited to direct DNA hybridization, hybridization of DNA to RNA or RNA to RNA, enzymatic assays such as polyemerase extension, ligation, . The microparticles can also be used in microfluidic or lab-on-a-chip systems or any flow based systems, including but not limited to those systems wherein sample preparation, biochemical reaction, and bio-analyses are integrated.

[00178] For example, fluorescent tags can be employed when an optical imaging method based on the presence of fluorescence can be used. Radioactive labels can be used when the microparticles are utilized to expose or develop relevant photographic films. Alternatively, enzymatic tags can be used when the detection involves detection of the product of the enzyme tag that is released when the sample molecules bind to or react with the probe molecules on the microparticles. Other tagging methods are also possible, as set forth in "Quantitative monitoring of gene expression patterns with a complementary DNA microarray" by Schena et al. Science, 1995, 270-467, the subject matter of which is incorporated herein by reference in its entirety.

[00179] Samples without labels can also be reacted with the microparticles. For example, molecular beacon probes can be applied to the microparticle. Molecular beacon probes typically contains a hairpin structure that, upon binding the labelless, or in some examples labeled, sample molecules unfold, thus producing a signal indicative of the binding events. Such molecular beacon probes, as well as other probes, may be used in assays involving FRET (Fluorescence Resonant Energy Transfer), where for example fluorophores or quenchers are placed on or in the surface of the microparticles.

Fig 48 shows real assay data from a 2-plex DNA hybridization assay. In this experiment, 2 different oligo probes (with 2 different sequences shown at the bottom) were attached to the surface of the different particle batches (with different codes). After probe attachment, the particles were mixed together and aliquots of the mixture were placed into two wells of a wellplate. Targets composed of oligos with sequences complementary to the probe sequences and fluorophore labels were then added to the two wells and reacted with the mixture of particle-probe conjugates. Targetl, complementary to probel, was added to the first well and target2, complementary to probe2, was added to the second well. Imaging of the particles of both wells was performed and the results are shown in Fig 48. In the first well (with targetl), particles of the corresponding code exhibit a relatively high fluorescence signal, and vice-versa for the second well.

[00180] For facilitating fast, reliable, and efficient bioassay for large number of sample molecules, it is preferred that the microparticles are capable of arranging themselves substantially in a monolayer on a surface, such as the bottom surface of the well in which the microparticles are contained. The microparticles are preferred to be able to undergo Brownian motion in the specific liquid in which the optical detection is performed. Given the specific liquid in which the microparticles are hybridized and detected, it is preferred that the 2D diffusion coefficient of the microparticles is equal to or greater than 1x10^'12 cm²/s and/or 10% or more, such as 15% or more, or even 20% or more, and 50% or more of the microparticles are measured to undergo a lateral displacement of 20 nm or greater, such as 3 Oram or greater, or even 50 ran or greater - in a time interval of 1 second or less, or preferably 3 seconds or less, or five seconds or less.

The detectable microparticles, which are referred to as those that are able to be accurately detected by the desired detection means, such as optical imaging using visible light, are capable of occupying 30% or more, 40% or more, and typically 50% or more of the surface area on which the microparticles are collected together, such as a portion of the bottom surface of the container in which the microparticles are contained. Defining an area in which at least 90% of all the microparticles are disposed (typically at least 95% or more typically at least 99%, and often 100%), the microparticles can be seen to have a density of 1000 particles/mm² or more, such as 1500 particles/Wn² or more, 2000 particles/Wz² or more, and typically 3000 particles/mm² or more (e.g. 5000 particles/mm² or more) . The detection rate within the above-mentioned area, which rate is defined as the ratio of the total number of detected microparticles (microparticles with spatial codes detected) of a collection of microparticles under detection to the total number of the collection of microparticles, is preferably 80% or more, typically 90% or more, or more typically 99% or more.

[00181] Another preferred example of the invention is a kit comprising biochemically active encoded microparticles that contains 200 or more, more preferably 500 or more, 1000 or more, or even 10,000 or more different codes within the kit (due to the large codespace enabled by the invention, even larger numbers of codes.) Due to statistical sample requirements of convenient liquid pipetting and a desired redundancy of particular codes within the kit, more than 10 particles of the same code are typically provided (20 or more, or even 30 or more microparticles of the same code) within the kit, as in some example applications the redundancy improves the overall assay performance. The term "biochemically active encoded microparticles" is refers to microparticles that have biological or chemical moieties on surfaces and thus can be used in assays; and the term "moieties" are referred to as molecular species; including but are not limited to nucleic acids, synthetic nucleic acids, oligonucleotides, single stranded nucleic acids, double stranded nucleic acids, proteins, polypeptides, antibodies, antigens, enzymes, receptors, ligands, and drug molecules, cells, and complex biologically derived samples.

[00182] Universal adapter schemes may be used to provide a set of non-interacting synthetic sequences that are complementary to sequences provided on the probes. Genotyping can be performed using common probes and allele specific reporters or allele specific probes and common reporters. Amplification assays such as those involving PCR, padlock probes, or Molecular Inversion Probes can be performed using the particles of the current invention. Examples of two of these assays are shown in FIG. 49A and FIG. 49B. In an alternative example of the invention, biomolecules that are present on the surface of the particles can be pre-synthesized and then attached to the particle surface. Alternatively, biomolecules can be in situ synthesized on the particles.

[00183] Protein based assays are also applicable. These include but are not limited to sandwich immunoassays, antibody-protein binding assays, receptor-ligand binding assays, or protein-protein interaction assays. Examples of these assays are shown in FIG. 50. The sets of encoded microparticles of the present invention can be used in solution based assays to investigate protein-protein interactions. This is shown in the bottom right of FIG. 50. [00184] A single type of protein can be applied to microparticles of a single code. Upon mixing of the particle-protein conjugates and reaction in a particular biochemical environment, proteins that interact and bind to one another are determined by the presence of adjacent particles during detection. The square cross section of the microparticle structures of the present invention provide an improvement over the prior art by providing an increased area of contact in the shape of a flat, rectangular surface. Prior art particles that are spherical or cylindrical in shape limit the contact areas to single points or lines respectively. This invention is not limited to proteins: any interacting molecules may be used with this assay architecture. Also, the omni-directional encoded microparticles of the present invention may be used in conjunction with any other encoded particles including but not limited to fluorophores, quantum dots, latex or glass beads, colloidal metal particles, spectroscopically active particles, SERS particles, or semiconductor nanorods.

[00185] The encoded microparticles may be used in conjunction with a 2D planar array of molecules. Interaction between molecules on the surface of the particles and those contained in spots on the 2D planar array are determined by the binding of the particles to the spots. The presence of the particles in the predetermined spot locations, preferably after washing steps, indicates a binding interaction between the molecules on the particles and the molecules on the 2D planar array. The assay result can be determined by identifying 1) the particle code, and 2) the spot location. This is shown in FIG. 51. FIG. 51 is a schematic that includes images of particles but is not the result of an actual experiment, i.e. meant to serve as an illustration of this invention. In this invention, the square cross section of the microparticles of the present invention provide for increased binding contact area and is a significant improvement over the prior art. [00186] The microparticles of the invention may have other applications. For example, by placing protein-detection molecules (e.g., ligands, dyes which change color, fluoresce, or cause electronic signal upon contact with specific protein molecules) onto the microparticles. , bioassay analyses can be performed (i.e., evaluation of the protein and/or gene expression levels in a biological sample). As another example, by placing (cellular) receptors, nucleic acids/probes, oligonucleotides, adhesion molecules, messenger RNA (specific to which gene is "turned on" in a given disease state), cDNA (complementary to mKNA coded-for by each gene that is "turned on"), oligosaccharides & other relevant carbohydrate molecules, or cells (indicating which cellular pathway is "turned on", etc.) onto the microparticles, the microparticles can be used to screen for proteins or other chemical compounds that act against a disease (i.e., therapeutic target); as indicated by (the relevant component from biological sample) adhesion or hybridization to specific spot (location) on the microarray where a specific (target molecule) was earlier placed/attached. In fact, the microparticles of the invention can be applied to many other biochemical or biomolecular fields, such as those set forth in the appendix attached herewith, the subject matter of each is incorporated herein by reference. [00187] It will be appreciated by those of skill in the art that a new and useful microparticle and a method of making the same have been described herein. The large sets of encoded microparticles produced by this invention can be a fundamental technology that will have far reaching applications, especially in the field of biotechnology and more specifically genomics. It has the potential to dramatically reduce the cost of highly multiplexed bioassays. Moreover, enables researchers to easily design custom content solution arrays. The researcher can also easily add new particle types to the pooled set, for instance including new found genes of interest with the microparticles of the invention.

[00188] In view of the many possible embodiments to which the principles of this invention may be applied, however, it should be recognized that the embodiments described herein with respect to the drawing figures are meant to be illustrative only and should not be taken as limiting the scope of invention. Those of skill in the art will recognize that the illustrated embodiments can be modified in arrangement and detail without departing from the spirit of the invention.

[00189] For example, the microparticle may have a six sided shape with four elongated sides and two end sides. The encoded microparticle can be configured such that the code of the encoded microparticle can be detectable regardless of which of the four elongated sides the barcode is disposed on. The microparticle may have a ratio of the length to width is from 2: 1 to 50:1, from4:l to 20:1. The length ofthemicroparticle is preferably from 5 to 100 um and more preferably less than 50 um. The width of the microparticle can be from 0.5 to 10 um. In other examples, the length of the microparticle can be less than 10 um, less than 25 um, less than 25 um; less than 5 um, less than 27 um; and the width of the microparticle can be less than 3 um. The ratio of width to height of the microparticle can be from 0.5 to 2.0. The ratio of the length to width of the microparticle can be from 2:1 to 50:1. The cross section taken along the length of the microparticle is substantially rectangular with a length at least twice the width. [00190] The microparticle may have a glass body with segments embedded therein. The difference of the transmissivity of the glass body and segments can be 10% or more. The glass body may have a length of less than 50 um and a width of less than 10 um with the glass body having a volume of from 5 to 500 um³. The encoded microparticle may have 2 to 15, 3 to 10, or 4 to 8 portions of less transparent material within the encoded microparticle. The code incorporated in the microparticle can be binary or non-binary or any other desired codes. The microparticle may have biochemical molecules attached to one or more surfaces of the microparticle, such as DNA and RNA probes with a density of from 10² tol0⁶/um². When fabricated on the wafer-level, the wafer may have a surface area of from 12.5 in² to 120 in², and wherein there are at least 3 million microparticles per in² of the wafer. The wafer may have at least one million codes are formed on the substrate, or at least two hundred different codes are present within the one million codes, or at least 3000 different codes are present within the one million codes. When placed in a liquid buffer, for example in a bioassay, the microparticles can form a single monolayer with a 2 dimensional diffusion coefficient of the microparticles greater than IxIO^"12 cm²/s and more preferably greater than IxIO^"11 cm²/s. [00191] Therefore, the invention as described herein contemplates all such embodiments as may come within the scope of the following claims and equivalents thereof.

APPENDIX

The subject matter of each one of the following references is incorporated herein by reference in entirety:

Preparing Probe Immobalized Arrays

G. Steinberg, K. Stromsborg., et al. Strategies for Covalent Attachment of DNA to Beads. Biopolymers. Vol. 73, 597-605. Published online 17 February, 2004 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20006.

Kumar, O. Larsson, D. Parodi, & Z. Liang. 2000. Silanized nucleic acids: a general platform for DNA immobilization. Nucleic Acids Research, 2000, Vol. 28. No. 14.

F. Vianello, L. Zennaro, et. al. Preparation, Morphological Characterization, and Activity of Thin Films of Horseradish Peroxidase. Biotechnology and Bioengeering, Vol. 68, NoI 5., June 5, 2000.

U. Maskos, & E. M. Southern. 1992. Oligonucleotide hybridixations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesized in situ. Nucleic Acids Research, Vol. 20, No. 7 1679-1684.

M. BeIe, O. Silman, & E. Matijevic. 2002. Preparation and Flow Cytometry of Uniform Silica-Fluorescent dye microspheres. Journal of Colloid and Interface Science. 254, 274-282.

N. Zammatteo, L. Jeanmart, et al. Comparison between Different Strategies of Covalent Attachment of DNA to Glass Surfaces to Build DNA Microarrays, Analytical Biochemistry. 280, 143-150 (2000)

N.P. Mellott, S. L. Brantley, et al. Evaluation of surface preparation methods for glass. Surface and interface Analysis. 2001; 31: 362-368

F. Dlehl, S. Grahlmann, et al. Manufacturing DAN microarrays of high spot homogeneity and reduced background signal. Nucleic Acids Research, 2001, Vol. 29, No. 7 e38.

Particle Arrays

Nicewarner-Pena, S.R., R.G. Freeman, B.D. Reiss, L. He, DJ. Pena, LD. Walton, R. Cromer, CD. Keating, and MJ. Natan, "Submicrometer Metallic Barcodes," Science, 294(5540), 137-141 (2001).

Natan, MJ., J. Lissack, "Barcoding Tackles the Nanometer," Tags and Lables ??

Walton, I.D., S.M. Norton, A. Balasingham, L. He, D.F. Oviso, D. Gupta, P.A. Raju, MJ. Natan, and R. G. Freeman, "Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy," Anal. Chem., 74, 2240-2247 (2002). True, RJ., M.K. Taylor, G.S. Chakarova, LD. Walton, "Microfabricated templates for the electrodeposition of metallic barcodes for use in multiplexed bioassays," IEEE - EMB Proceedings, 26(IV), 2619-2622 (2004).

Xu, H.X., M.Y. Sha, E.Y. Wong, J. Uphoff, Y.H. Xu, J.A. Treadway, A. Truong, E. O'Brien, S. Asquith, M. Stubbins, et. al., "Multiplexed SNP genotyping using the Qbead (TM) system: a quantum dot-encoded microsphere-based assay," Nucleic Acids Res., 31(8), E43 (2003).

Han, M., X. Gao, J.Z. Su, S. Nie, "Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules," Nat. Biotechnol., 19(7), 631-635 (2001).

Protein Arrays

Haab, B.B., M.J. Dunham, & P.O. Brown. 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Geneome Biology. 2(2): 0004.1-0004.13.

MacBeath, G., & SX. Schreiber. 2000. Printint proteins as microarrays for high-throughput function determination. Science. 289; 1760-1763.

Zhu, H., J. F. Klemic, et al.2000. Analysis of yeast protein kinases using protein chips. Nature Genetics. 26: 283-289.

Zhu, H., & M. Snyder. 2001. Protein arrays and microarrays. Current Opinion in Chemical Biology. 5: 40-45.

B. B. Haab, M. J. Dunham & P. O. Brown. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biology 2001, 2(2) research 0004.1-0004.13

Glycan Arrays

Blixt, O;Head, S;Mondala, T;Scanlan, QHuflejt, ME;Alvarez, R;Bryan, MQFazio,

FjCalarese, D;Stevens, J;Razi, N;Stevens, DJ;Skehel, JJ;van Die, I;Burton, DR; Wilson,

IA;Cummings, R;Bovin, N; Wong, CH;Paulson, JC.2004 Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. PROC NAT ACAD SCI USA 101:49

17033-17038

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome. Comelli, E. M. et al. Glycobiology 16(2), 117-31, 2006

Glycomics investigation into insulin action. Parry, S. et al. Biochimica Et Biophysica Acta-General Subjects 1760(4), 652-68, 2006

Cancer Chips

Alizadeh. A.A., M. B. Eisen, et al. 2000. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 403: 503-511.

Ein-Dor, L., I. KeIa, et al. 2005. Outcome signature genes in breast cancer: is there a unique set? Bioinformatics. 21(2): 171-178. Ross,D.T., U. Scherf, et al. 2000. Systematic variation in gene expression patters in human cancer cell lines. Nature Genetics. 24: 227-238.

Sorlie, T., R. Tibshirani, et al. 2003. Repeated observation of breast tumor subtypes in independent gene expression data sets. PNAS USA. 200: 8418-8423.

Aneuploidy Chips

Behr, M. A., M.A. Wilson, et al. 1999. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science. 284: 1520-1523.

Forozan, F., E. H. Mahlamaki, et al. 2000. Comparative genomic Hybridization anaysis of 38 breast cancer cell lines: A basis for interpreting complementary DNA microarray data. Cancer Research. 60: 4519-4528.

Grimm, D. 2004. Disease back cancer origin theory. Science 306: 389.

Hughes, T.R., C. J. Roberts, et al. 2000. Widespread aneuploidy revealed by DNA microarray expression profiling. Nature Genetics. 25: 333-337

Leptin Chips

Belkin, L. 2000, December 24. The making of an 8-year-old woman. How do we understand puberty? Through the prism of our times. New York Times magazine. 38-43.

Nohturfft, A., & R. Losick. 2002. Fats, flies and palmitate. Science. 296: 857-858.

Soukas, A., P. Cohen, et al. 2000. Leptin-specific patterns of gene expression in white adipose tissue. Genes and Development. 14: 963-980.

Microarray Fabrication Methods

Brown, P.O., et al. 2001. The Mguide. http://cmgm.stanford.edu/pbrown/mguide/. Accessed 8 February 2002.

Kane, M.D., T.A. Jatkoe, et al. 2000. Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Research. 28(22): 4552-4557.

Kumar, A., & A. Liang. 2001. Chemical nanoprinting: A novel method for fabricating DNA microchips. Nucleic Acids Research 29(2): e2.

Okamoto, T., T. Suzuki, & N. Yamamoto. 2000. Microarray fabrication with covalent attachment of DNA using bubble jet technology. Nature Biotechnology. 18: 438-441.

Harris, T. M., A. Massimi, & G. Childs. 2000. Injecting new ideas into microarray printing. Nature Biotechnology. 18:384-385.

General References Cummings, C.A., & D. Relman. 2000. Using DNA microarrays to study host-microbe interactions. Genomics. 6(5): 513-525.

Schaffer, J.L., M. Perez- Amador, & E. Wisman, 2000. Monitoring genome-wide expression in plants. Current Opinion in Biotechnology. 11: 162-167

Lockhart, D. J., & C. Barlow. 2001. Expressing what's on your mind: DNA arrays and the brain. Nature Reviews. 2: 63-68.

Shoemaker, D. D., E. E. Schadt, et al. 2001. Experimental annotation of the human genome using microarray technology. Nature. 409: 922-927.

Dalton, R. 2000. DIY (Do it yourself) microarrays promise DNA chips with everything. Nature. 403: 236

DeRisi, J. L. V. R. Iyer, & P. O. Brown 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science. 278:680-686.

Gasch, A. P., P. T. Spellman, et al. 2000. Genomic expression programs in the response of yeast cells to environmental changes. Molecular Biology of the Cell. 11: 4241-4257

Geschwind, D. H. 2000. Mice, microarrays, and the genetic diversity of the brain. PNAS. 97: 10676-10678.

R. Drmanac, S. Drmanac, G. Chui, et al. Sequencing by Hybridization (SBH): Advantages, Achievements, and Opportunities. Advanced in Biochemical Engineering/Biotechnology, Vol. 77, 75-100.

Membrane Chips

Fang Y, Frutos AG, Webb B, Hong Y, Ferrie A, Lai F, Lahiri J. Membrane biochips. Biotechniques. 2002 Dec;Suppl:62-5. PMID: 12514931

Fang Y, Frutos AG, Lahiri J. G-protein-coupled receptor microarrays. Chembiochem. 2002 Oct4;3(10):987-91. PMID: 12362364

Fang Y, Frutos AG, Lahiri J. Membrane protein microarrays. J Am Chem Soc. 2002 Mar 20;124(ll):2394-5. PMID: 11890761

Fang Y, Webb B, Hong Y, Ferrie A, Lai F, Frutos AG, Lahiri J. Fabrication and application of G protein-coupled receptor microarrays. Methods MoI Biol. 2004;264:233-43. PMID: 15020794

Fang Y, Lahiri J, Picard L. G protein-coupled receptor microarrays for drug discovery. Drug Discov Today. 2003 Aug 15;8(16):755-61. Review. PMID: 12944098

Mishina YM, Wilson CJ, Bruett L, Smith JJ, Stoop-Myer C, Jong S, Amaral LP, Pedersen R, Lyman SK, Myer VE, Kreider BL, Thompson CM. Multiplex GPCR assay in reverse transfection cell microarrays. J Biomol Screen. 2004 Aρr;9(3): 196-207. PMID: 15140381 Mack J. Fulwyler and Thomas M. McHugh, "Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes," Methods In Cell Biology, vol. 33, pp. 613-619.

Fulwyler et al, "Immunorealtive Bead (IRB) Assay for the Quantative and Simultaneous Flow Cytometric Detection of Multiple Analytes", Cytometry, Supplement 2, p. 19, Sep. 2, 1988.

Fulwyler et al., "Flow Micropheres Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes," Methods in Cell Biology, 33, 613-629 (1990).

Vener et al., "A Novel Approach to Nonradioactive Hybridization Assay of Nucleic Acids Using Stained Latex Particles," Analytical Biochemistry, 198, 308-311 (1991).

Wilson et al., "A New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry," Journal of Immunological Methods, 107, 225-230 (1988).

Bayerl, T. M. & Bloom, M. Physical properties of single phospholipid bilayers adsorbed to micro glass beads. Biophys. J. 58, 357-362 (1990).

Buranda, T. et al. Biomimetic molecular assemblies on glass and mesoporous silica microbeads for biotechnology. Langmuir 19, 1654-1663 (2003).

Pathogen Detection

Seung Min Yoo, Ki Chang Keum, et al. 2004. Development of DNA Microarray for Pathogen Detection. Biotechnology and Bioprocess Engineering. 9: 93-99

Call DR, Borucki MK, Loge FJ. Detection of bacterial pathogens in environmental samples using DNA microarrays. J Microbiol Methods. 2003 May;53(2):235-43. Review. PMID: 12654494

Somer L, Kashi Y. A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products. J Food Prot. 2003 Sep;66(9): 1658-65. PMID: 14503721

Warsen AE, Krug MJ, LaFrentz S, StanekDR, Loge FJ, Call DR. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays. Appl Environ Microbiol. 2004 Jul;70(7):4216-21. PMID: 15240304

Maynard C, Berthiaume F, Lemarchand K, Harel J, Payment P, Bayardelle P, Masson L, Brousseau R. Waterborne pathogen detection by use of oligonucleotide-based microarrays. Appl Environ Microbiol. 2005 Dec;71(12):8548-57. PMID: 16332846

Gonzalez SF, Krug MJ, Nielsen ME, Santos Y, Call DR. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J Clin Microbiol. 2004. Apr;42(4): 1414-9. PMID: 15070982

M. T. McBride, S. M. Messenger, T. R. Slezak, & P. M. Imbro. Tailored assays for the detection of foreign disease pathogens in animals. IVD Technology, May 2005 SMALL RNA

Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs "Guillier, M. et al. Molecular Microbiology 59(1), 231-47, 2006

Genes for small, noncoding RNAs under sporulation control in Bacillus subtilis Silvaggi, J. M. et al. Journal of Bacteriology 188(2), 532-41, 2006

Comparative RNA expression analyses from small-scale, single-donor platelet samples Hillmann, A. G. et al. Journal of Thrombosis and Haemostasis 4(2), 349-56, 2006

Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells

Shou, H. Y. et al Journal of Pharmacological and Toxicological Methods 53(2), 152-9, 2006

An antibody-based microarray assay for small RNA detection Hu, Z. L. et al. Nucleic Acids Research 34(7), NPG, 2006

Notch3 gene amplification in ovarian cancer

Park, J. T. et al. Cancer Research 66(12), 6312-8, 2006

Dissecting the biological functions of Drosophila histone deacetylases by RNA interference and transcriptional profiling

Foglietti, C. et al. Journal of Biological Chemistry 281(26), 17968-76, 2006 c-Myb is an essential downstream target for homeobox-mediated transformation of hematopoietic cells

Hess, J. L. et al. Blood 108(1), 297-304, 2006

Localization of candidate regions for a novel gene for Kartagener syndrome Gutierrez-Roelens, I. et al. European Journal of Human Genetics 14(7), 809-15, 2006

MicroRNA-targeted and small interfering RNA-mediated rnRNA degradation is regulated by Argonaute, Dicer, and RNA-dependent RNA polymerase in Arabidopsis Ronemus, M. etal. Plant Cell 18(7), 1559-74, 2006

Methylatioπ

High-Throughput DNA Methylation Profiling Using Universal Bead Arrays, M. Bibikova, Z. Lin, L. Zhou, E. Chudin, E. Wickham Garcia, B. Wu, D. Doucet, N. J. Thomas, Y. Wang, E. Vollmer, T. Goldmann, C. Seifart, W. Jiang, D. L. Barker, M. S. Chee, J.A. Floros and J.B. Fan, Genome Research, 16 (3), 383 - 393, March 2006.

Human Embryonic Stem Cells Have a Unique Epigenetic Signature, M. Bibikova, E. Chudin, B. Wu, L. Zhou, E. Wickham Garcia, Y. Liu, S. Shin, T. W. Plaia, J. M. Auerbach, D. E. Arking, R. Gonzalez, J. Crook, B. Davidson, T. C. Schulz, A. Robins, A. Khanna, P. Sartipy, J. Hyllner, P. Vanguri, S. Savant-Bhonsale, A. K. Smith, A.Chakravarti, A. Maitra, M. Rao, D. L. Barker, J. F. Loring and J. B. Fan, Genome Research, Published online August 9, 2006, 10.1101/gr.5319906. Tagging

Summary of the Sensing and Positioning Technology, Workshop of the Committee on nanotechnology for Intelligence Community: Interim Report (2004), National Materials Advisory Board, Topic 1 : Security Technologies Overview

Desouza, K.C. Vanapalli, G.K. Securing Knowledge Assets and Processes: Lessons from the Defense and Intelligence Sectors System Sciences, 2005. HICSS '05. Proceedings of the 38th Annual Hawaii International Conference on

Publication Date: 03-06 Jan. 2005

Books

D. Bowtell and J. Sambrook. 2003, DNA Microarrays, A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press (in particular, sections 1-4)

G.T. Hermanson, Bioconjugate Techniques, 1996, Academic Press (Parts 1, 2 and 3 are all directly applicable)

Hacia J, Brody L,Chee M, et at "Detection of heterozygous mutations in BRCAl using high density oligonucleotide arrays and two-color fluorescence Analysis" Nat Genet, 1996, 14, 441.

Hacia J, Edgemon K,Sun B et al. "Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes" Nucleic Acids Res, 1998, 26, 4249.

Hacia J. "Resequencing and mutation analysis using oligonucleotides microarrays" Nature Genetics (Supplement), 1999, 21, 42.

Wodicka L et at "Genome-wide expression monitoring in Saccharomyces cerevisiae" Nature Biotechnology, 1997, 15, 1359.

Lockhart D et at "Expression monitoring by hybridization to high-density oligonucleotide arrays" Nature Biotechnology, 1997, 14, 1675.

DeRisi J. et at "Use of a cDNA microarray to analysis gene expression patterns in human cancer" Nat Genet, 1996, 14, 457.

Brown P O, Botstein D. "Exploring the new world of genome with DNA microarrays" Nature Genetics (Supplement), 1999, 21, 33.

Jelinsky S and Samson L. "Global response of Saccharomyces cerevisiae to a alkylating agent" proc. Natl Acad Sci USA, 1999, 96, 486.

Golub T. et at, 1999, "Molecular Classification of cancer: Class Discovery and Prediction by Gene Expression Monitoring" Science, 1999, 286, 531.

Heller RA, Schena M, Chai A, et at "Discovery and analysis of inflammatory disease-related genes using cDNA microarrays" .Proc Natl Acad Sci USA, 1997, 94, 2150. Wang D, Fan J, Siao C et al. "Large-scale identification, mapping and genotyping of single-nucleotide polymorphism inhuman genome" Science, 1998, 280, 1077.

Lipshutz RJ, Morris D, Chee M, et al. "Using oligonucleotide probe arrays to access genetic diversity" Bio Feature, 1995, 19(3), 442.

Wallraff G., Labadie J., Brock P. et al. "DNA Sequencing on a Chip" Chemtech, 1997, 22, 32.

Claims

I Claim:

1. An encoded rqicroparticle, comprising; a first material comprising two or more separate segments aligned along an axis;

S a second material surrounding the first material such that the segments are detectable through the second material; and wherein a code for the microparticle is provided.

2, The microparticle of claim 1 , wherein the code of the microparticle is from a codespace mat is greater tirø* 1,000. 0 3. The microparticle of claim 1 , wherein the code of the microparticle is from codespace that is greater than 10,000.

4. The microparticle of claim 1, wherein the second material is transparent,

5. The microparticle of claim 1, wherein the first material is opaque. o\ The microparticle of claim 4, wherein the transparent material comprises glass. 5 7. The microparticle of claim 1 , wherein the first material is opaque.

8. The microparϋck of claim 7, whcieixi the entire outer sυrface of the inicroparticle is glass.

9. The microparticle of claim 1, wherein the first material comprises a semiconductor or metal.

10. The microparticle of claim 1 , wherein a cross section of the microparticle is substantially rectangular. 0 11, The π-icroparu^'cle of claim 1 , wherein a volume of the microparticle is 20,000 urn³ or less .

12. The micToparticle of claim 1, wherein a length of the microparticle is 50 urn or less.

13. The microparticle of claim 15, wherein a width of the microparticle is S urn or less.

14. The microparticle of claim 1, wherein the ratio of the length to the width of the microparticle is 3:1 or higher. 5 15. The microparticle of claim 1 , wherein the microparticle comprises a magnetic material.

16. The microparticle of claim 1, wherein the second material comprises a plurality of fluorescent molecules therein or thereon.

17. The microparticle of claim 1 , wherein the second material comprises a surface on which a multiplicity of biochemical molecules is attached. 0 18. The microparticle of claim 1, wherein the wiicroparticles are substantially disposed in a monolayer.

19, The microparticle of claim 1 , wherein the røicroparticles are disposed in a liquid and the microparticles undergo Brownian motion and have biochemical molecules attached to the surface.

20, The microparticle of claim 1 , wherein the second material comprises a plurality of indentations in a surface thereof, 5 21. A multiplicity of encoded microparticles, each microparticle comprising: a fust material comprising two or more separate segments aligned along an axis; a second material surrounding the first material such that the segments are detectable through the second material; and wherein a code for the microparticle is provided. 0 22. The multiplicity of encoded microparticles of claim 21, wherein the second material is transparent. 23. The multiplicity of encoded microparticles of claim 21, wherein the first material is opaque.

24. The multiplicity of encoded microparticles of claim 23, wherein the first material comprises a semiconductor or metal.

25. The multiplicity of encoded microparticles of claim 21, wherein a cross section of the microparticle is substantially rectangular . 26. The multiplicity of encoded microparticles of claim 21 , wherein a volume of the microparticle is 20,000 um¹ or less.

27. The multiplicity of encoded microparticles of claim 26, wherein a length of the microparticle is 50 urn or less.

28. The multiplicity Of encoded microparticles of claim 26, wherein a width of the microparticle is 5 urn or less.

29. The multiplicity of encoded microparticles of claim 26, wherein the ratio of the length to the width of the πύcropartifclβ is 2\\ at higher.

30. The multiplicity of encoded microparticles of claim 26, wherein the microparticle comprises a magnetic material, 31. The multiplicity of encoded microparticles of claim 21, wherein the second material comprises a plurality of fluorescent molecules therein oi thereon,

32. The multiplicity of encoded microparticles of claim 21, wherein the second material comprises a surface on which a multiplicity of biochemical molecules is attached.

33. A method of forming an encoded rnicroparticle, comprising: depositing and patterning a layer on a substrate so as to form a plurality of microparticks, each comprising a plurality of separate segments aligned along an axis and representing a code; and releasing the microparticles in order to separate the microparticles from the substrate.

34. The method of claim 33, wherein the step of depositing and patterning the layer further comprises: depositing a first layer on the substrate; depositing a second layer on the substrate; forming the second layer into a plurality of particle regions such that each particle region has two or more discrete segments aligned along the axis; and depositing a third layer on the substrate;

35. The method of claim 33, wherein the step of patterning the kyer comprises: forming, at a first time a first pattern on the layer; forming at a second time a second pattern on the layer; and wherein the first and second patterns form the mioroparticle.

36. The method of claim 34, wherein the first material is transparent.

37. The method of claim 34, wherein the second material is opaque. 38. The method of claim 37, wherein the second material comprises a semiconductor or metal.

39. The method of claim 34, wherein a cross section of the microparticle is substantially rectangular.

40. The method of claim 34, wherein a volume of the microparticle is 20,000 um' or less.

41. The method of claim 34, wherein a length of the microparticle is 50 um or less.

42. The method of claim 41, wherein a width of the mioroparticle is 5 um or less. 43. The method of claim 34, furthor comprising: forming a layer that is composed of a magnetic material. 44. The method of claim 34, further comprising: forming a fluorescent layer composed of a plurality of molecules.

45. The method of claim 34, farther comprising: attaching a multiplicity of biochemtoal molecules on or in a surface microparticle.

46. A method of producing an encoded particle comprising: S providing a substrate; depositing a first layer on the substrate; depositing a second layer on the substrate; forming the second layer into a plurality of particle regions such that each panicle region has two or more discrete segments aligned along an axis; 0 depositing a third layer on the substrate; releasing the layers in the particle regions from the substrate to form individual panicles; wherein a code is formed by the discrete segments of an individual particle.

47. The method of claim 46, wherein the step of patterning the layer comprises: forming, at a first time a first pattern on the layer; S fanning at a second time a second pattern on the layer; and wherein the first and second patterns form the micioparticle. 4$, The method of claim 47, wherein the first material is transparent.

49. The method of claim 47, wherein the second material is opaque.

50, The method of claim 47, wherein the second material comprises a semiconductor or metal. 0 51. The method of claim 47, wherein a cross section of the microparticle is substantially rectangular.

52. The method of claim 47, wherein a volume of the microparticle is 20,000 urn³ or less.

53. The method of claim 47, wherein a length of the rnicroparticle is 50 urn or less.

54. The method of claim 47, wherein a width of the microparticle is 5 urn or less,

55. The method of claim 47, further comprising: 5 forming a layer that i$ composed of a magnetic material.

56. The method of claim 47, further comprising: forming a fluorescent layer composed of a plurality of molecules.

57. The method of claim 47, further comprising: attaching a multiplicity of biochemical molecules on or in a surface microparticle 0 58. A method of forming an encoded microparticle, comprising: depositing and patterning a layer on a substrate so as to form a plurality of microparticle., each comprising a plurality of discrete segments aligned along an axis which is substantially parallel to the substrate and representing a code; and releasing the microparticles in order to separate the microparticle. from the substrate, wherein the code for 5 each microparticle is detectable from all directions perpendicular to the axis. 59. The method of claim 58, wherein patterning the layer comprises; forming, at a first time a first pattern on the layer; forming at a second time a second pattern on the layer; and wherein the first and second patterns form the microparticle, 0 60. The method of claim 58, wherein the first material is transparent.

61. The method of claim 58, whereto the second material is opaque.

62. The method of claim 58, wherein the second material comprises a semiconductor or metal.

63. The method of claim 58, wherein a cross section of the raicroparticle is substantially rectangular.

64, The method of claim 58, wherein a volume of the micrαparticle is 20,000 um³ or less,

65. The method of claim 58, wherein a length of the microparticle is 50 um or leas.

66, The method of claim 58, wherein a width of the microparticle is 5 um or less. S 67. The method of claim 58, further comprising: forming a layer that is composed of a magnetic material.

68. The method of claim 58, further comprising; forming a fluorescent layer composed of a plurality of molecules.

69. The method of claim 58, further comprising: 0 attaching a multiplicity of biochemical molecules on or in a surface microparticle.

70. An encoded microparticle comprising: a first materia! comprising two or more discrete segments aligned along an axis; a second material that is transparent and is surrounding the first material such that the segments are detectable through the second material} and 5 wherein a code for the microparticle is provided and is detectable from all angles orthogonal to the axis.

71. The method of claim 70, wherein the step of patterning the layer comprises: forming, at a first time a first pattern on the layer; forming at a second time a second pattern on the layer; and wherein the first and second patterns form the microparticle. 0 72. The method of claim 70, wherein the first material is transparent,

73. The method of claim 70, wherein the second material is opaque.

74. The method of claim 73, wherein the second material comprises a semiconductor or metal,

75. The method of claim 71, wherein a cross section of the microparticle is substantially rectangular.

76. The method of claim 75, wherein a volume of the microparticlB is 20,000 um³ or less. 5 77. The method of claim 75, wherein a length of the microparticle is 50 um or less.

78. The method of claim 75, wherein a width of the micropartiole is S um or less.

79. The method of claim 75, further comprising: forming a layer that is composed of a magnetic material

80. The method of claim 75, further comprising; 0 forming a fluorescent layer composed of a plurality of molecules.

81. The method of claim 75 , further comprising: attaching a multiplicity of biochemical molecules on or in a surface microparticle.

82. An encoded microparticle comprising: an elongated body comprising a plurality of alternating portions of a more transparent material and a less 5 transparent material, wherein the portions of the less transparent material are adjacent to the portions of more transparent materials; and wherein the portions of the more and less transparent materials represent a detectable code.

83. The encoded microparticle of claim 82, wherein the less transparent material is opaque.

84. The encoded microparticle of claim 83, wherein the more transparent material ia glass. 0 85. The encoded microparticle of claim 83, wherein the less transparent material is a more absorbing material in the visible spectrum.

86. The encoded microparticle of claim S3, wherein the less transparent material is a more light reflecting material.

87. The encoded microparticle of claim 83, wherein the opaque material comprises a semiconductor or metal,

88. The encoded microparticle of claim 83, wherein a cross section of the microparticle is substantially rectangular.

89. The encoded microparticle of claim 83, wherein a volume of the microparticle is 20,000 um³ or less.

90. The encoded microparticle of claim 83, wherein a length of the microparticle is 50 um or less.

91. The encoded microparticle of claim 90, wherein a width of the microparticle is 5 um or less.

92. The encoded microparticle of claim 90, wherein an aspect ratio of the mioroparticle is 3:1 or higher. 93. The encoded microparticle of claim 90, wherein the microparticle comprises a magnetic material.

94. The encoded microparticle of claim 83, wherein the elongated body comprises a plurality of fluorescent molecules therein or thereon.

95. The encoded microparticle of claim 83, wherein the elongated body comprises a surface on which a multiplicity of biochemical molecules is attached. 96. A method of detecting an encoded microparticle, comprising: providing a microparticle having a spatial code along an axis and having a plurality of elongated sides; providing incident light onto the microparticle; detecting light reflected or transmitted through the miciopartitfe; wherein the code of the microparticle can be detected independent of which of the elongated sides the light is incident on.

97. The method of claim 96, wherein the light has a spectrum in a visible or near-visible light range.

98. The method of claim 96, wherein the microparα^'cte comprises: a first material comprising two or more separate segments aligned along an axis; a second material surrounding the first material such that the segments are detectable through the second material; and wherein a code for the microparticle Is provided.

99. The method of claim 98, wherein the microparticle is a member of a codespace of the microparticles and the codespace is greater than 1000.

100. The method of claim 98, wherein the microparticle is a member of a codespace of the microparticles and the codespace is greater than 10000.

101. The method of claim 99, wherein the second material is transparent.

102. The method of claim 99, wherein the first material is opaque.

103. The method of claim 99, wherein a cross section of the raicropaπicle is substantially rectangular.

104. The method of claim 99, wherein a volume of the microparticle is 20,000 um³ or less, 105. The method ofdairn 99, wherein a length of the microparticle is 50 um or less.

106, The method of claim 105, wherein a width of the microparticle is 5 um or less.

107. The method of claim 105, wherein the ratio of the length to the width of the microparticle i. 3:1 or higher. 103, The method of claim 105, wherein the second material comprises a plurality of fluorescent molecules therein or thereon. 109. The method of clairo 105, wherein the second material comprises a surface on which a multiplicity of biochemical molecules is attached. 110. The method of claim 99, wherein the microparticles arc substantially disposed in a monolayer.

111. The method of claim 110, wherein the microparticles are disposed in a liquid buffer and the microparticles undergo Brownian motion and have biochemical molecules attached to the surface.

112. The method of claim 105, wherein the second material comprises a plurality of indentations in a surface thereof. 113. An encoded microparticle comprising: a longest dimension less than SOum; an outer surface substantially of glass; and a spatial code that can be read with optical magnification.

114. The microparticle of claim 113, wherein the spatial code is detectable with reflected light. 1 IS. The microparticle of claim 113, wherein the spatial code is detectable with transmitted light

116. The microparticle of claim 113, wherein the spatial code is detectable with emitted light.

117. The microparticle of claim 113, wherein the spatial code is detectable with a single image capture event.

118. The microparticle of claim 113, wherein the microparticle has a volume of 20,000 um³ or less. 115», The microparticle of claim 113, wherein the microparticle has a volume of 5000 urn³ or less. 120. The microparticle of claim 113, wherein the microparticle has a volume of 1000 urn³ or less.

121, The nxicroparticle of claim 113, wherein the code of the microparticle is ftom codespace that is greater than 1,000.

122. The microparticle of claim 113, wherein .the code of the mlcropartiole is from codespace that is greater than 10,000. 123. The microparticle of claim 113, wherein the microparticle comprises an opaque material.

124. The microparticle of claim 123, wherein the opaque material comprises a semiconductor or metal.

125. The micrσparticle of claim 113, wherein a cross section of the microparticle is substantially rectangular,

126. The microparticle of claim 113, wherein the ratio of the length to the width the ratio of the length to width of the microparticle is 3:1 or higher. 127. The microparticle of claim 113, wherein the microparricle comprises a magnetic material.

128. The microparticle of claim 113, wherein the second material comprises a plurality of fluorescent molecules therein or thereon,

129. The microparticle of claim 113, further comprising a multiplicity of biochemical molecules on a surface of the microparticle. 130. The microparticle of claim 113, wherein the microparticle is a member of a plurality of microparticles that is substantially disposed in a monolayer.

131. The microporticle of claim 130, wherein the microparticles are disposed in a liquid buffer and the microparticles undergo Brownian motion and have biochemical molecules attached to the surface.

132. The microparticle of claim 130, further comprising a plurality of indentations in a surface of the microparticle.

133. The microparticle of claim 113, wherein a length of the microparticle is 50 urn or less,

134. The microparticle of claim 133, wherein a length of the microparticle is 20 urn or less.

135. An encoded microparticle comprising: a longest dimension less than 50umj an outer surface substantially of glass; and a spatial code comprising code elements with a maximum size of 1.5 urn or less.

136. The microparticle of claim 135, wherein the maximum size is 1.0 um or less.

137. The microparticle of claim 135, wherein the microparticle has a volume of 20000 tun⁹ or less.

138. The microparticle of claim 135, wherein the microparticle has a volume of 5000 um' or less.

139. The microparticle of claim 135, wherein the microparticle further comprises: a first material comprising two or more separate segments aligned along an axis; a second material surrounding the first materia] such that the segments are detectable through the second material; and wherein a code for the microparticlc is provided.

140. The microparticle of claim 139, wherein the spatial code is detectable with a reflectance imaging system.

141. The microparticle of claim 139, wherein the spatial code is detectable with a transmissive imaging system. 142. The micropaiticle of claim 139, wherein the spatial code is detectable with a fluorescence imaging.

143. The microparticle of claim 139, wherein the spatial code is detectable with a single image capture event.

144. The microparticle of claim 135, wherein the microparticle comprises an opaque material.

145. The microparticle of claim 135» wherein a cross section of the microparticle is substantially rectangular.

146. The mioroparticle of olaim 135, wherein the ratio of the length to the width of the microparticle is 3:1 or higher.

147. The microparticle of claim 135, wherein the microparticle comprises a magnetic material.

148. The microparticle of claim 135, further comprising a plurality of fluorescent molecules on or in all or a potion surface of the microparticle,

149. The microparticle of claim 135, further comprising a multiplicity of biochemical molecules on. a surface of the microparticle.

150. The microparticle of claim 135, wherein the microparticle is a member of a plurality of microparticles that is substantially disposed in a monolayer.

151. The microparticle of claim 150, wherein the microparticles are disposed in a liquid and the microparticles undergo Brownian motion and have biochemical molecules attached to the surface. 152. The microparticle of claim 151, further comprising a plurality of indentations in a surface of the microparticle.

153. The microparticle of claim 135, wherein the spatial code is detectable with reflected light.

154. The microparticle of claim 135, wherein the spatial code is detectable with transmitted light.

155. The microparticle of claim 135, wherein the spatial code is detectable with emitted light. 156. The microparticle of claim 135, wherein the microparticle comprises a transparent material.

157. The microparticle of claim 135, further comprising a plurality of fluorescent molecules on a surface of the microparticle.

158. A method of forming a plurality of encoded microparticles with a printing process to define a code for identifying the particle comprising the steps of: a) providing a pattern that defines a single code element per microparticle region; b) printing a first code clement with the pattern; c) printing a successive code element with the pattern such that the first and successive code elements are within t-be same microparticle region; d) wherein codes on the microparticle is provided.

159. The method of claim 158, wherein the printing steps (b) or (c) are performed with photolithography.

160. The method of claim 1S8, wherein the printing comprises: we use of a step and repeate exposure system.

161. The method of claim 159, wherein the printing step is performed by exposure of a photosensitive layer.

162. The method of claim 158, wherein the printing steps of (b) or (c) is performed with imprint lithography.

163. The method of claim 158, wherein the pattern is provided on a reticle. 164. The method of claim 158, wherein the codes are formed of a plurality of separate and opaque segments,

165. The method of claim 158, wherein the substrate on which microparticles are formed is laterally shifted between exposure steps.

166. Λ method for forming a plurality of encoded microparticles, comprising: printing at a first time a first portion of the codes of the microparticles; and printing at a second time after the first time a second portion of the codes of the microparticles, such that the first portion and second portion form at least in part the code for each microparticle.

167. The method of claim 166, wherein either one of Hie printing seeps is performed with photolithography.

168. The method of claim 167, wherein the printing comprises: stepping snd repeating an exposure system. 169. The method of claim 167, wherein the printing step is performed by exposure of a photosensitive layer.

170. The method of claim 166, wherein either one of the printing steps is peτfgrøied with stamping.

171. The method of claim 166, wherein the pattern is provided on a reticle.

172. The method of claim 166, wherein the codes are formed of a plurality of alternating more transmissive and less trausmissive segments, 173. The method of claim 172, wherein the substrate on which microparticles are formed is laterally shifted between exposure steps,

174. A method of forming a plurality of encoded microparticles with a lithography process to define a codes for the microparticles for identifying the ^"microparticles, comprising: a) providing a substrate comprising a plurality of encoded microparticles; b) providing a pattern comprising identification elements; c) printing the pattern on the substrate or a portion of the substrate; d) laterally shifting the substrate by a predefined distance; e) executing c again; and f) wherein a code on the particle is provided. 175. The method of claim 174, wherein the printing step is performed with photolithography.

176. The method of claim 174, wherein the printing comprises : the use of a step and repeαtε exposure system,

177. The method of claim 176, wherein the printing step is performed by exposure of a photosensitive layer

178. The method of claim 174, wherein the encoded microparticles comprise an outer layer of glass. 179. The method of claim 176, wherein the pattern is provided on a reticle.

ISO. The method of claim 176, wherein the codes are formed of a plurality of separate and opaque segments.

181. The method of claim 177, wherein the substrate on which microparticles are formed is laterally shifted between exposure steps.

182, A system, comprising; a step and repeat exposure system capable of performing a method comprising: printing at a first time a first portion of a code of a microparticle; and printing at a second time after the first time a second portion of a code of the miorαparticle; and a computer readable medium having a sequence of computer executable instructions for controlling said step and repeat exposure system to perform said method,

183. A computer readable medium having computer executable instructions for performing a method, the method comprising: directing a printing system to produce a set of encoded microparticles, the set comprising a plurality of regions, each region comprising a plurality of encoded microparticles all having the same code, the computer program comprising a list of coordinate locations and lateral offsets that define the codes of the different regions.

184. A system, comprising: a step and repeat exposure system capable of performing a method comprising: printing at a first time a first portion of a code of a mlcroparticle; and printing at a second time after the first time a second portion of a code of the microparticle; and a computer readable medium having a sequence of computer executable instructions for controlling said step and repeat exposure system to perform said method.

155. A method of forming a plurality of encoded microparticle with a lithography process to define a code for identifying the microparticles comprising the steps of: a) providing a substrate; b) depositing a first layer; c) depositing a second layer; d) forming the microparticle codes according to instructions of a step end repeat exposure system; e) wherein codes on the particle are provided.

186. The method of claim 185, wherein the step of forming the particle regions further comprises; providing a substrate; providing a pattern comprising identification elements; printing the pattern on the substrate or a portion of the substrate; laterally shifting the substrate by a predefined distance; executing c again; and wherein a code on the particle is provided.

187. The method of claim 186, wherein the printing step is performed withphotolithography.

188. The method of claim 186, wherein the printing comprises: the use of a step and repeate exposure system.

189. The method of claim 186, wherein the printing step is performed by exposure of a photosensitive layer,

190. The method of claim 186, wherein the printing step ia embossing lithography.

191. The method of claim 186, wherein the pattern is provided on a photomask.

192. The method of claim 186, wherein the codes are formed of a plurality of separate and opaque segments. 193. The method of claim 189, wherein the substrate on which microparticles are formed is laterally shifted between exposure steps.

194. A method for forming encoded micioparticles, comprising: depositing on a substrate a first layer to be patterned; depositing on the layer to be patterned A second layer that is a photoresist layer; with a step and repeat exposure system, forming an exposure pattern in the photoresist layer by stepping a code element pattern across the photoresist layer; selectively removing the photoresist layer and patterning the first layer; wherein the step and repeat exposure system is programmed to form different microparticle codes on the same substrate; and separating the microparticlcs from the substrate.

195. The method of claim 194, farther comprising:

S depositing another layer before depositing the first layer.

196. The method of claim 194, wherein the printing comprises: stepping and repeating an exposure system.

197. The method of claim 194, wherein the the code element pattern is provided on a reticle.

198. The method of claim 194, wherein the codes are formed of a plurality of separate and opaque segments. 0 199. The method of claim 194, wherein the substrate on which microparticles are formed is laterally shifted between exposure steps.

200. The method of claim 197, wherein the code element pattern comprises a subset of the exposure pattern.

201. The method of claim 197, wherein the code element pattern is provided on a reticle.

202. The method of claim 197, wherein the distance that the substrate is laterally shifted by an amount that is 5 less than the longest dimension of a particle.

203. A microparticle, comprising: an elongated body having an axis, wherein a cross-section taken perpendicular to the axis is substantially square.

204. An encoded microparticle comprising: 0 an elongated structure comprising a detectable spatial code, where the elongated structure has a substantially square cross section; and the elongated structure further comprises a material opaque to visible or near visible light and a material transmissive to the visible or near visible light.

205. The microparticle of claim 204, wherein the opaque material is enclosed within the transmissive material. 5 206. The microparticle of claim 204, wherein the opaque material forms an opaque segment fully surrounded by the transmissive material

207. The microparticle of claim 2OS, wherein the light transmissive material is glass,

208. The microparticle of claim 207, wherein the light opaque material oomprises silicon.

209. The microparticle of claim 207, wherein the glass is in a form of a solid body with the opaque segment 0 embedded therein such that the minimum thickness of the glass surrounding the segment is 5 microns.

210. The microparticle of claim 209, wherein the minimum thickness is 17 microns,

211. An encoded microparticle comprising: a cuboid with an aspect ratio of a major surface orthogonal to the length of the microparticle greater than

3:1; 5 a substantially square cross section taken orthogonal to the length of the microparticle; and opaque and transparent portions,

212. The microparticle of claim 211, wherein the opaque and transparent portions alternate along the length of the microparticle so as to represent a code.

213. The microparticle of claim 212, wherein the opaque segment is embedded within the microparticle such 0 that substantially all surfaces of the segment are enclosed by the exterior surfaces of the microparticle without contact.

214. The microparticle of claim 213, wherein the opaque portion is composed of amorphous silicon.

215. The microparticle of claim 213, wherein the transparent portion is composed of glass.

216. The microparticle of claim 215, wherein the glass comprises silicon oxide.

217. The microparticle of claim 216, wherein the microparticle has a maximum dimension of 50 microns.

218. An encoded microparticle comprising: a particle body having a length, width and height, wherein the length is from 5 to 100 miorons, wherein the width and height are from 0.5 to 10 microns, and wherein the ratio of width to height is from 0.5 to 2.0.

219. The microparticle of claim 218, wherein the ratio of width to height is substantially 1.

220. The microparticle of claim 218, further comprising: a set of segments and spacers intervening the segments disposed along the length of the microparticle for representing a code derived from a predetermined coding scheme.

221. The microparticle of claim 219, wherein the segments are composed of a material that is opaque to visible or near-visible light; and wherein the spacers are composed of a material having a transmisalvity of 50% or more to the visible or near visible light.

222. A wafer comprising: a plurality of discrete die areas, each die area having a plurality of encoded microparticles that are patterned but unreleased from the wafer; and wherein the wafer has a surface area of from 12.5 in¹ to 120 in², and wherein there are at least 3 million microparticles per in² of the wafer.

223. The wafer of claim 222, wherein each microparticle is composed of a fist portion of a first material and a second portion of a second material.

224. The wafer of claim 222, wherein the first material is more transmissive to visible or near-visible light than the second material,

225. The wafer of claim 224, wherein the more transparent material comprises silicon dioxide.

226. The wafer of claim 224, wherein the more transparent material is an organic material. 227. The wafer of claim 224, wherein the more transparent material fully encapsulates the portions of the less transparent material.

228. The wafer of claim 224, wherein the more transparent material comprises silicon nitride.

229. The wafer of claim 224, wherein the detectable code is detectable under τβflectBd light from the microparticle. 230. The wafer of claim 224, wherein the detectable code is detectable under transmitted light passing through the microparticle.

231. The wafer of claim 224, wherein the less transparent material comprises silicon.

232. The wafer of claim 224, wherein the less transparent material comprises amorphous silicon.

233. The wafer of claim 224, wherein the microparticle is a six sided shape with four elongated sides and two end sides.

234. The wafer of claim 233, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

235. The wafer of claim 224, further comprising a biological material immobilized on a surface of the more transparent material. 236. The wafer of claim 235, wherein the biological material comprises at least a portion of a nucleoside. 237. The wafer of claim 224, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

238. The wafer of claim 237, wherein the intermediate portions of more transparent materia! have lengths defined βs the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle,

239. The wafer of claim 224, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square,

240. The wafer of claim 224, wherein from 2 to 15 portions of less transparent material arc provided within the encoded microparticle.

241. The wafer of claim 240, wherein frora 4 to 8 portions of less transparent material are provided within the encoded microparticle. 242. The wafer of claim 240, wherein the portions of less transparent material have varying lengths.

243. The wafer of claim 224, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

244. The wafer of claim 224, wherein each microparticle has a size such that a collection of the microparticle when released is capable of undergoing Brownian motion in a buffer. 245. The wafer of claim 224, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

246. The wafer of claim 224, wherein the less transparent material comprises polysilicon. .

247. The wafer of claim 224, wherein the alternating portions of more and less transparent material form a non- binary code. 248. The wafex of claim 224, wherein the alternating portions of more and less transparent material form a binary code.

249. The wafer of claim 236, wherein the nucleoside is a DNA or RNA probe immobilized on a surface of the encoded microparticle at a density of from 10¹ tol0^β/um^J

250. The wafer of claim 249, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

251. The wafer of claim 224, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween.

252. The wafer of claim 224, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon. 253. The wafer of claim 252, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

254. The wafer of claim 224, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

255. The wafer of claim 254, wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon,

256. The wafer of claim 222, wherein the microparticle comprises a ratio of the length to width is from 2: 1 to 50:1.

257. The wafer of claim 222, wherein the microparticle comprises a length of less than 50 um and a width of less than 10 um. 258. The wafer of claim 222, wherein the microparticle comprises a volume of from 5 to 500 um³. 259. A wafer comprising: a plurality of die areas each having a plurality of unreleased microparticles; wherein each microparticle comprises a code formed from sequentially arranged materials that can be optically differentiated from each other; and wherein the number of die areas multiplied by the number of particles in each die area divided by the diameter of the wafer in centimeters is from AA to BB. 260. The wafer of claim 259, wherein each microparticle is composed of a fist portion of a fust material and a second portion of a second material.

261 , The wafer of claim 259, wherein the first material is more transmissive to visible or near-visible light than the second material.

262. The wafer of claim 261, wherein the more transparent material comprises silicon dioxide, 263. The wafer of claim 261, wherein the more transparent material is an organic material.

264. The wafer of claim 261, wherein the more transparent material fully encapsulates the portions of the less transparent material.

265. The wafer of claim 261, wherein the more transparent material comprises silicon nitride.

266. The wafer of claim 261, wherein the detectable code is detectable under reflected light from the microparticle.

267. The wafer of claim 261 , wherein the detectable code is detectable under transmitted light passing through the microparticle.

268. The wafer of claim 261, wherein the less transparent material comprises silicon.

26°. The wafer of claim 261, wherein the less transparent material comprises amorphous silicon. 270. The wafer of claim 261, wherein the microparticle is a six sidod shape with four elongated sides and two end sides.

271. The wafer of claim 270, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

272. The wafer of claim 264, further comprising a biological material immobilized on a surface of the more transparent material.

273. The wafer of claim 272, wherein the biological material comprises at least a portion of a nucleoside.

274. The wafer of claim 264, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

275. The wafer of claim 274, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

276. The wafer of claim 264, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

277. The wafer of claim 264, wherein from 2 to 15 portions of less transparent material are provided within the encoded microparticle.

278. The wafer of claim 277, wherein from 4 to 8 portions of less transparent material axe provided within the encoded microparticle.

279. The wafer of claim 277, wherein the portions of less transparent material have varying lengths.

280. The wafer of claim 264, wherein the potions of losa transparent material have substantially -the same lengths whereas the portions of more transparent material have varying lengths,

281. The wafer of claim 264, wherein each microparticle has a size such that a collection of the microparticle when released is capable of undergoing Brownian motion in a buffer.

282. The wafer of claim 264, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

283. The wafer of claim 264, wherein the less transparent material comprises polysilicon.

284. The wafer of claim 264, wherein the alternating portions of more and less transparent material form a non- binary code.

285. The wafer of claim 264, wherein the alternating portions of more and less Transparent material form a binary code.

286. The wafer of claim 273, wherein the nucleoside is a DNA or KNTA probe immobilized on a surface of the encoded microparticle at a density of from HO² to,0⁶/um². 287. The wafer of claim 286, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

288. The wafer of claim 264, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween.

289. The wafer of claim 264, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon.

290. The wafer of claim 289, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

291. The wafer of claim 264, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon. 292. The wafer of claim 291 , wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon.

293. The wafer of claim 264, wherein the microparticle comprises a ratio of the length to width is from 2:1 to 50:1.

294. The wafer of claim 264, wherein the microparticle comprises a length of less than 50 urn and a width of less than 10 um.

295. The wafer of claim 264, wherein the microparticle comprises a volume of from 5 to 500 um³.

296. A silicon wafer comprising a plurality of iraxeleased glass microparricles Ihereon, wherein the number of microparticles on the wafer is from YY to ZZ per cm².

297. The wafer of claim 296, wherein the number of microparticles on the wafer is from YY' to ZZ' per 1 cm². 298. The wafer of claim 296, further comprising XXXX or more die areas each of which formed thereon a plurality of microparticles.

299. The wafer of claim 298, wherein each die area comprises XXXXXX or more micropam^'cles.

300. The wafer of claim 296, wherein each πύcroparticle is composed of a first portion of a first material and a second portion of a second material. 301. The wafer of claim 296, wherein the first material is more transmissive to visible or near-visible light than the second material.

302. The wafer of claim 300, wherein the more transparent material comprises silicon dioxide.

303. The wafer of claim 300, wherein the more transparent material is an organic material.

304. The wafer of claim 300, wherein the more transparent material fully encapsulates the portions of the less transparent material.

305. The wafer of claim 300, wherein the more transparent material comprises silicon nitride.

306. The wafer of claim 300, wherein the detectable code is detectable under reflected light from the microparticle.

307. Tie wafer of claim 300, wherein the detectable code is detectable under transmitted light passing through the micioparticle, 5 308. The wafer of claim 300, wherein the less transparent material comprises silicon.

309. The wafer of claim 300, wherein the less transparent material comprises amorphous silicon.

310. The wafer of claim 300, wherein the rαicroparticle is a six sided shape with four elongated sides and two end sides.

311. The wafer of claim 310, wherein the code of the encoded microparticle is detectable regardless of which of 10 the four elongated sides the barcode is disposed on.

312. The wafer of claim 300, further comprising a biological material immobilized on β surface of the more transparent material.

313. The wafer pfclaim 312, wherein the biological material comprises at least a portion of a nucleoside.

314. The wafer of claim 300, wherein each less transparent portion defines intermediate portions of more 15 transparent material between adjacent two less transparent portions.

315. The wafer of claim 314 wherein the intermediate portions of more transparent materiel have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

316. The wafer of claim 300, wherein a cross section taken orthogonal to the length of the encoded microparticle 20 is substantially square.

317. The wafer of claim 300, wherein from 2 to 15 portions of less transparent material are provided within the encoded microparticle.

318. The wafer of claim 317, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle.

25 319. The wafer of claim 300, wherein the portions of less transparent material have varying lengths.

320. The wafer of claim 300, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

321. The wafer of claim 300, wherein each microparticle has a size such that a collection of the microparticle when released is capable of undergoing Brownian motion in a buffer.

30 322. The wafer of claim 300, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

323. The wafer of claim 300, wherein the less transparent material comprises polysilicon.

324. The wafer of claim 300, wherein the alternating portions of more and less transparent material form a non- binary code.

J5 325. The wafeir of claim 300, wherein the alternating portions of more and less transparent material form a binary code.

326. The wafer of claim 312, wherein the nucleoside is a DNA or RNA ptobe immobilized on a surface of the encoded micropartiele at a density of from IQ² toloVum².

327. The wafer of claim 326, Vvhβrein the DNA or RNA probe is bound to at least a portion of a complementary 40 biochemical material from a sample with a fluorescent tag.

328. The wafer of claim 300, wherein the portions of the less transparent material are discrete and separate segments disposed with a jacent portions of the more transparent material therebetween.

329. The wafer of claim 300, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon,

330. The wafer of claim 329, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon. 331 , The wafer of claim 300, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

332. The wafer of claim 331, wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon.

333. The wafer of claim 300, wherein the πήcroparticle comprises a ratio of the length to width is from 2:1 to 50:1.

334. The wafer of claim 300, wherein the microparticle comprises a length of less than 50 um and a width of less than 10 urn.

335. The wafer of claim 300, wherein the microparticle comprises a volume of from 5 to 500 um³.

33d. A silicon wafer having a plurality of unreleased encoded microparticles formed thereon, comprising: a plurality of encoded microparticles bonded to the silicon; the microparticles having substantially the entire outer surface comprised of silicon dioxide, with a lower portion of the outer surface directly bonded to the silicon wafer.

337. The wafer of claim 336, wherein each microparticle is composed of a fi$t portion of a first material and a second portion of a second material. 338. The wafer of claim 336, wherein the first material is more transmissive to visible or near-visible light than the second material.

339. The wafer of claim 337, wherein the more transparent material comprises silicon dioxide.

340. The wafer of claim 337, wherein the more transparent material is an organio material.

341. The wafer of claim 337, wherein the more transparent material fully encapsulates the portions of the less transparent material.

342. The wafer of claim 337, wherein the more transparent material comprises silicon nitride.

343. The wafer of claim 337, wherein the detectable code is detectable under reflected light from the microparticle.

344. The wafer of claim 337, wherein the detectable code is detectable under transmitted light passing through the microparticle.

345. The wafer of claim 337, wherein the less transparent material comprises silicon.

346. The wafer of claim 337, wherein the less transparent material comprises amorphous silicon.

347. The wafer of claim 337, wherein the microparticle is a six sided shape with four elongated sides and two end sides. 348. The wafer of claim 347, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

349. The wafer of claim 337, further comprising a biological material immobilized on a surface of the more transparent material.

350. The -wafer of claim 349, wherein the biological materiel comprises at least a portion of a nucleoside. 351. The wafer of claim 337, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

352. The wafer of claim 351 , wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

353. The wafer of claim 337, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

354. The wafer of claim 337, wherein from 2 to 15 portions of less transparent material are provided within the encoded πύcioparticle,

355. The wafer of claim 353, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle. 356. The wafer of claim 337, wherein the portions ofless transparent material have varying lengths.

357. The wafer of claim 337, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

358. The wafer of claim 337, wherein each microparticle has a size such that a collection of the rrricropartide when released is capable of undergoing Brownian motion in a buffer. 359. The wafer of claim 337, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

360, The wafer of claim 337, wherein the less transparent material comprises pαlysiUcon,

361. The wafer of claim 337, wherein the alternating portions of more and less transparent material form a non- binary code. 362. The wafer of claim 337, wherein the alternating portions of more and less transparent material form a binary code.

363. The wafer of claim 361, wherein the nucleoside is a DNA or RNA probe immobilized on a surface of the encoded raicroparricle at a density of from 10^z tolOVum².

364. The wafer of claim 363, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

365. The wafer of claim 337, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween,

366. The wafer of claim 337, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon. 367. The wafer of claim 366, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

368. The wafer of claim 337, wherein the portions of the less transparent material reflect at least S0% of the visible light Incident thereon,

369. The wafer of claim 363, wherein the portions of the less transparent material reflect at least 60% of the visible light incident thereon.

370. The wafer of claim 337, wherein the uiicroparticle comprises a ratio of the length to width is from 2; 1 to 50:1.

371. The wafer of claim 337, wherein the microparticle comprises a length of less than 50 urn and a width of less than 10 um. 372. The wafer of claim 337, wherein the microparticle comprises a volume of from 5 to 500 um^]. 373. A substrate having unreleased encoded microparticles comprising: a plurality of encoded mioroparticles connected to the substrate in the absence of an intervening sacrificial layer, the microparticles having an outer surface comprised of glass. •

374. The substrate of claim 373, wherein the substrate is a single Crystal silicon.

375. The substrate of claim 373, wherein the microparticles have a length and width measured parallel to the substrate surface and a height measured perpendicular to the substrate surface such that the width to height ratio is less than or equal to 5:1.

376. The substrate of claim 373, wherein the microparticle has a shape of a solid rectangular parallelepiped.

377. The substrate of claim 373, wherein a plurality of raicroparticles foimed on a top surface of the silicon wafer, wherein the microparticles comprise a bottom layer of silicon dioxide bound to the top surface of the silicon wafer, and a top layer of silicon dioxide material, with an intermediate layer between the top and bottom silicon dioxide layers that forms a detectable spatial binary or non-binary code.

378. The substrate of claim 377, wherein each micioparticle is composed of a fist portion of a first material and a second portion of a second material, 379. The substrate of claim 373, wherein the first material is more transmissive to visible or near-visible light than the second material.

380. The substrate of claim 379, wherein the more transparent material comprises silicon dioxide.

381. The substrate of claim 379, wherein the more transparent material is an organic material.

382. The substrate of claim 379, wherein the more transparent material fully encapsulates the portions of the less transparent material.

383. The substrate of claim 379, wherein the more transparent material comprises silicon nitride.

384. The substrate of claim 379, wherein the detectable code is detectable under reflected light from the microparticle.

385. The substrate of claim 379, wherein the detectable code is detectable under transmitted light passing through the microparticle.

386. The substrate of claim 379, wherein the less transparent material comprises silicon.

387. The substrate of claim 379, wherein the less transparent material comprises amorphous silicon.

388. The substrate of claim 379, wherein the microparticle is a six sided shape with four elongated sides and two end sides. 389. The substrate of claim 386, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

390. The substrate of claim 379, further comprising a biological material immobilized on a surface of the more transparent material.

391. The substrate of claim 388, wherein the biological material comprises at least a portion of a nucleoside, 392. The substrate of claim 379, wherein each les$ transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

393, The substrate of claim 390, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions axe substantially the saroβ regardless of location within the encoded microparticle. 394, The substrate of claim 379, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

395. The substrate of claim 379, wherein from 2 to IS portions of less transparent material are provided within the encoded microparticle.

396. The substrate of claim 393, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle. 397. The substrate of claim 379, wherein the portions of less transparent material have varying lengths.

398. The substrate of claim 379, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

399. The substrate of claim 379, wherein each microparticle has a βize such that a collection of the microparticle when released iβ capable of undergoing Brownian motion in a buffer. 400. The substrate of claim 379, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

401. The substrate of claim 379, wherein the less transparent material comprises polysilicon.

402. The substrate of claim 379, wherein the alternating portions of more and less transparent material form a non-binary code. 403. The substrate of claim 379, wherein the alternating portions of more and less transparent material form a binary code.

404. The substrate of claim 389, wherein the nucleoside is a DNA or KNA probe immobilized on a surface of the encoded microparticle »t a density of from 10² tolO^δ/um².

405. The substrate of claim 402, wheiein the DNA oτ RNA probe is bound to at least a portion of a complementary biochemical material fiom a sample with a fluorescent tag.

406. The substrate of claim 379, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween.

407. The aubstrate of claim 379, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon. 408. The substrate of claim 405, wherein the portions of the le.s transparent material absorb at least 80% of the visible light incident thereon,

409. The substrate of claim 379, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

410. The substrate of claim 407, wherein the portions of (he less transparent material reflect at least 80% of the visible light incident thereon.

411. The substrate of claim 379, wherein the microparticle comprises a ratio of the length to width is from 2:1 to 50:1.

412. The substrate of claim 379, wherein the microparticle comprises a length of less than 50 um and a width of less than 10 um. 413. The substrate of claim 379, wherein the microparticle comprises a volume of from 5 to 500 um³.

414. The wafer of claim 379, wherein the intermediate layer is a patterned optically absorptive or reflective layer that is of a material that is different from the silicon dioxide layers and can be optically differentiated from the silicon dioxide layers,

415. The wafer of cl&im 379, wherein the patterned intermediate layer is patterned as a series of discrete segments within a single microparticle.

416. The wafer of claim 413, wherein the discrete segments of the intermediate layer comprise silicon dioxide between adjacent segments.

417. The wafer of claim 414, wherein the plurality of microparticles formed on a top surface of the semiconductor wafer, wherein the micrgparticles comprise a bottom layer of a first material substantially transmisaive to light in the visible spectrum, wherein the first material is bound to the top surface of the semiconductor wafer, and wherein a top layer of a second material, that can be the same or different from the first

S material, that is substantially transmissive to light in the visible spectrum, and further comprising an intermediate layer between the top and bottom substantially traπsπussive layers, which intermediate layer comprises a spatial pattern that forms a detectable binary or non-binary code.

418. A method con-prising, providing the semiconductor wafer of claim 336; 0 singulating the wafer into individual dies each having a semiconductor wafer portion and microparticles thereon,- placing the individual dies into separate containers; etching the semiconductor wafer portions in each container so as to undercut the attached rnicroparticles and separate the micro-particles therefrom. 5 419, The method of claim 418, wherein a liquid etchant is provided in each container or well.

420. The method of claim 418, wherein the liquid is TMAH.

421. The method of claim 418, wherein the containers are a plurality of wells in a multiple well plate.

422. The method of claim 418, wherein the number of wells is from XXXX to YYYY.

423. A method comprising: 0 providing a wafer that comprises a plurality of micxopaiticlea formed on a top surface of the wafer, wherein the microparticles comprise a spatial pattern that forms a detectable binary or non-binary codej singulating the wafer into individual dies; providing the individual dies into separate containers along with an etchant; etching the wafer underneath the plurality of rnicroparticles so as to release the microparticles from the 5 wafer.

424. The method of claim 423, wherein the wafer is a silicon wafer and the etchaxrt etches into the surface of the silicon wafer in an amount that rally undercuts and releases the particles.

425. The method of claim 424, wherein a liquid etchant is provided in each container or well.

426. The method of claim 424, wherein the liquid is TMAH. 0 427. The method of claim 424, wherein the containers are a plurality of wells in a multiple well plate.

428. The method of claim 423, wherein the number of wells is from XXXX to YYYY.

429. The method of claim 423, wherein the wafer comprises 20 or more dies each of which comprises a plurality of microparticles.

430. The method of claim 423, wherein the wafer comprises 50 or more dies each of which comprises a plurality 5 of microparticles.

431. The method ofclaim 430, wherein each dies comprises 100 or more microparticles,

432. The method ofclaim 431, wherein each die comprises 10000 or more microparticles.

433. The method of claim 431, wherein the wafer comprises 500,000 or more microparticles,

434. The method of claim 431 , wherein the wafer comprises 1 ,000,000 or more microparticles, 0 435. The method ofclaim 429, wherein the microparticles in the same die have the same oode.

436. The method of claim 429, wherein the raioroparticles in different dies have different codes,

437. The method of claim 423, wherein the different codes are derived from a pre-deiermined coding scheme.

438. A method comprising: providing a substrate that comprises a plurality of inicropartioles foπned on a top surface of the substrate, wherein the microparticlej comprise a spatial pattern that forms a detectable binary or non-binary code; providing the substrate into a container along with an etchant; and bulk etching the substrate underneath the plurality of microparticles so as to release the micropg∑ticles from the wafer.

439. The method of claim 438, further comprising: providing a silicon wafer or silicon wafer portion; forming a plurality of glass microparticles each comprising a spatial code on a top surface of the silicon wafer; bulk etching the silicon wafer or silicon wafer portion in order to release the microparticles therefrom.

440, The method of claim 439, wherein the wafer is a silicon wafer and the etchant etches into the surface of the silicon wafer in an amount that fully undercuts and releases the particles. '

441, The method of claim 439, wherein a liquid etchant is provided in each container or well. 442. The method of claim 439, wherein the liquid is TMAH.

443. The method of claim 441, wherein the containers are a plurality of wells in a multiple well plate,

444. The method of claim 439, wherein the number of wells is from XXXX to YYYY.

445. The method of claim 439, wherein the wafer comprises 20 or more dies each of which comprises a plurality of microparticles. 446, The method of claim 439, wherein the wafer comprises 50 or more dies each of which comprises a plurality of microparticles,

447, The method of claim 446, wherein each dies comprises 100 or more microparticles.

448, The method of claim 447, wherein each die comprises 10000 or more microparticles.

449, The method of claim 447, wherein the wafer comprises 500,000 or more microparticles, 450, The method of claim 449, wherein the wafer comprises 1 ,000,000 or more microparticles.

451. The method of claim 439, wherein the microparticles in the same die have the same code.

452. The method of claim 439, wherein the microparticles in different dies have different codes.

453. The method of claim 452, wherein the different codes are derived from a pre-determined coding scheme.

454. A method of forming a plurality of encoded microparticles comprising; a) providing a substantially planar substrate; b) forming a plurality of encoded microparticles on the surface of the substrate; c) releasing the encoded microparticles by bulk etching the substrate so as to undercut the microparticles and detach the microparticles from the substrate.

455. The method of claim 454, wherein the wafer is a silicon wafer and the etchant etches into the surface of the silicon wafer is an amount that fully undercuts and releases the particles.

456. The method of claim 455, wherein a liquid etchant is provided in each container or well.

457. The method of claim 456, wherein the liquid is TMAH.

458. The method of claim 456, wherein the containers are a plurality of wells in a multiple well plate.

459. The me&αd of olnim 457, wherein the number of wells is from XXXX to YYYY. 460. The method of claim 457, wherein the wafer comprises 20 or more dies each of which comprises a plurality of microparticles.

461. The method of claim 457, wherein the wafer comprises 50 or more diea each of which comprises a plurality ofrnicroparticles,

462. The method of claim 457, wherein each dies comprises 100 or more microparticles.

463. The method of claim 457, wherein each die comprises 10000 or more microparticles. 464. The method of claim 457, wherein the wafer comprises 500,000 or more microparticles.

465. The method of claim 457, wherein the wafer comprises 1 ,000,000 or more microparticles.

466. The method of claim 457, wherein the microparticles in the same die have the same code.

467. The method of claim 457, wherein the microparticles in different dies have different codes.

468. The method of claim 457, wherein the different codes are derived from a pre-determined coding scheme. 469. A method of forming a plurality of encoded microparticles comprising: a) providing a substrate; b) depositing a silicon sacrificial layer of silicon on the substrate; c) forming a plurality of encoded microparticles on the surface of the silicon sacrificial layer; d) releasing the encoded microparticles by etching the silicon sacrificial layer with an etchant that etches silicon,

470. The method of claim 469, wherein the wafer is a silicon wafer and the etchant etches into the surface of the silicon wafer in an amount that fully undercuts and releases the particles

471. The method of claim 469, -wherein a liquid etchant is provided in each container or well.

472. The method of claim 469, wherein the liquid is TMAH. 473. The method of claim 469, wherein the containers are a plurality of wells in a multiple well plate,

474. The method of claim 469, wherein the number of wells is from XXXX to YYYY.

475. The method of claim 469, wherein the wafer comprises 20 or more dies each of which comprises n plurality of microparticles.

476. The method of dlairn 469, wherein the wafer comprises 50 or more dies each of which comprises a plurality of microparticles.

477- The method of claim 476, wherein each dies comprises 100 or more microparticles.

478. The method of claim 477, wherein each die comprises 10000 or more microparticles.

479. The method of claim 477, wherein the wafer comprises 500,000 or more microparticles.

480. The method of claim 477, wherein the wafer compri$es 1 ,000,000 or more microparticles. 481 , The method of claim 477, wherein the microparticles in the same die have the same code.

482. The method of claim 477, wherein the micropaiticles in different dies have different codes,

483 ■ The method of claim 482, wherein the different codes are derived from a pre-deterrained coding scheme.

484. A wafer comprising: a plurality of silicon dioxide mioroparticles bonded to a top surface of the wafer, wherein the top surface of the wafer is a silicon surface.

485. A method for releasing microparticles, comprising: providing a silicon wafer with a plurality of microparticleθ thereon; bulk etching the silicon wafer so as to undercut the microparticles and detach the nαicroparticles from the silicon wafer, 486, A microparticle comprising: a particle body having a plurality of indentations in a surface of the elongated body, wherein on or in the surface of the particle body is provided a fluorescent materials; wherein the fluorescent material appears as areas of brighter and darker portions; where the brighter portions correspond to the indentations in the surface of the elongated body; and wherein a spatial code is detectable based on the brighter and darker portions of fluorescent material.

487. The microparu^'cle of claira486, wherein the particle body comprises a firstpoition of a more transπήssive S material and a second portion of a less transmisBive material to visible or near-visible light.

483, The microparticle of claim 487, wherein the more transparent material comprises silicon dioxide.

489. The microparticle of claim 487, wherein the silicon dioxide is a thin film CVD material.

490. The microparticle of claim 487, wherein the more transparent material is an organic material,

491. The microparticle of claim 487, wherein the more transparent material is a hybrid organic-inorganic 10 material.

492. The microparticle of claim 487, wherein the more transparent material fully encapsulates the portions of the less transparent material.

493. The microparticle of claim 487, wherein the more transparent material is a ceramic compound.

494. The microparticle of claim 493, wherein the ceramic compound comprises an oxide of a metalloid or early 15 transition metal.

495. The microparticle of claim 493, wherein the ceramic compound comprises a nitride of a metalloid or early transition metal.

496. The microparticle of claim 493, wherein (he ceramic compound comprises a carbide of a metalloid or early transition metal.

SO 497. The microparticle of claim 496, wherein the more transparent material comprises silicon nitride.

498. The microparticle of claim 487, wherein the detectable code is detectable under reflected light from the microparticle, said light having a wavelength in the visible and/or near visible spectrum.

499. The microparticle of claim 487, wherein the detectable code i. detectable under transmitted light passing through the mieroparticle, said light having a wavelength in the visible and/or near visible spectrum.

£5 500, The microparticle of claim 487, wherein the less transparent material comprises silicon.

501. The microparticle of claim 500, wherein the less transparent material comprises amorphous silicon,

502. The microparticle of claim 487, wherein the less transparent material is an opaque material under visible or near visible light.

503. The micropaiticle of claim 487, wherein the less transparent material comprises an early transition metal. 10 504, The microparticle of claim 487, wherein the less transparent material comprises a metalloid.

505. The microparticle of claim 504, wherein the less transparent material comprises aluminum.

506. The microparticle of claim 505, wherein the less transparent material comprises tungsten, chromium, titanium, tantalum or molybdenum.

507. The microparticle of claim 487, wherein the less transparent material comprises silicon or aluminum, and 15 the more transparent material comprises silicon dioxide.

508. The microparticle of claim 487, having a six sided shape with four elongated sides and two end sides.

509. The microparticle of claim 508, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

510. The microparticle of claim 487, further comprising a biological material immobilized on a surface of the (0 more transparent material.

511. The microparticle of claim 510, wherein the biological material comprises a segment of DNA.

512. The microparticle of claim 487, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

513. The rrricroparticle of claim 512, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the

5 intermediate portions are substantially the same regardless of location within the encoded microparticle.

514. The microparticle of claim 508, wherein a cross section taken orthogonal to the length of the encoded tnicropartrøle is substantially square,

515. The πήcroparticle of claim 487, wherein from 2 to 15 portions of leas transparent material are provided within the encoded microparticle.

0 516. The microparticle of claim S15, wherein from 3 to 10 portions of less transparent material are provided within the encoded microparticle

517. The microparticle of claim SlS, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle.

518. The microparticle of claim 515, wherein the portions of less transparent material have varying lengths.

.5 519. The microparticle of claim 487, wherein the potions of lesB transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

520. The microparticle of claim 487, wherein the portions of more transparent material and the portions of less transparent material all have varying lengths.

521. The microparticle of claim 487, that has a size that undergoes Browπian motion when in a buffer.

!0 522. The microparticle of claim 487, wherein the more transparent material is formed of a spin-on material.

523. The microparticle of claim 487, wherein the more transparent material is a βiloxane or silsesquioxane material,

524. The microparticle of claim 487, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous.

!5 525. The microparticle of claim 487, wherein the portions of less transparent material are fully surrounded by the more transparent material.

526. The microparticle of claim 487, wherein the less transparent material comprises polysilicon.

527. The microparticle of claim 487, disposed within a buffer in a well of a multi-well plate,

528. The micropartiele of claim 487, wherein the alternating portions of more and less transparent material form 10 a non-binary code,

529. The microparticle of claim 487, wherein the alternating portions of more and less transparent material form a binary code.

530. The microparticle of claim 510, wherein the biological material comprises a segment of RNA.

531. The microparticle of claim 510, wherein the biological material is a DNA or UNA probe immobilized on a S5 surface of flie encoded microparticle at a density of from 10² toloVum*.

532. The microparticle of claim 531, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

533. The microparticle of claim 487, wherein the less transparent material comprises a magnetic material.

534. The microparticle of claim 533, wherein the magnetic material comprises nickel, cobalt or iron.

(0 535, The micropartielo of claim 487, wherein ⁽he portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween.

536. The microparticle of claim 487, wherein the portions of toe less transparent material absorb at least 50% of the visible light incident thereon.

537. The microparticle of claim 536, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon. 538. The microparticle of claim 487, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

539. The microparticle of claim 536, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

540. The microparticle of claim 487, wherein a ratio of the length to width is from 2: 1 to 50:1. 541. The microparticle of claim 540, wherein the ratio ofthc length to width is from 4:1 to 20;l.

542. The microparticle of claim 517, having a length of less than 50 urn and a width of less than 10 um

543. The microparticle of claim 548, having a length of less than 25 υm and a width of less than 5 um.

544. The microparticles of claim 543, having a length of less than 27 um and a width of less than 3 um.

545. The microparticle of claim 487, having a volume of from 5 to 500 um³. 546. The microparticle of claim 545, having a volume of from 10 co 200 um³.

547. A microparticle comprising: a particle body with luminescent material on or in the surface thereof; and a plurality of surface features providing a varying aerial density of luminescence that forms a code.

548. The microparticle of claim 547, wherein the particle body comprises a first portion of a more transmissive material and a second portion of a less bansmissive materia! to visible or near-visible light.

549. The microparticte of claim 548, wherein Ae more transparent material comprises silicon dioxide.

550. The microparticle of claim 548, wherein the silicon dioxide is a thin film CVD material.

55 \ . The micioparticle of claim 548, wherein the more transparent material is an organic material.

552. The microparticle of claim 548, wherein the more transparent material is a hybrid organic-inorganic material.

553. The microparticle of claim 548, wherein the more transparent material fully encapsulates the portions of the less transparent material.

554. The microparticle of claim 54S₁ wherein the more transparent material is a ceramic compound.

555. The rπicroparticle of claim 553, wherein the ceramic compound comprises an oxide of a metalloid or early transition metal,

556. The microparticle of claim 553, wherein the ceramic compound comprises a nitride of a metalloid or early transition metal.

557. The microparticle of claim 553, wherein the ceramic compound comprises a carbide of a metalloid oτ early transition metal. 558. The microparticle of claim 555, wherein the more transparent material comprises silicon nitride.

559. The αdcropartide of claim 548, wherein the detectable code is detectable under reflected light from the microparticle, said light having a wavelength in the visible and/or near visible spectrum.

560. The miotoparticle of claim 548, wherein the detectable code is detectable under transmitted light passing through the tnicropaiticle, said light having a wavelength in the visible and/or near visible spectrum. 561. The microparticle of claim 548, wherein the less transparent material comprises silicon.

562. The microparticle of claim 561, wherein the less transparent material comprises amorphous silicon.

563. The microparticle of claim 548, wherein the less transparent rnaterial is an opaque material under visible or near visible light,

564. The microparticle of claim 548, wherein the less transparent material com,rises aa early transition metal.

565. The microparticle of claim S48, wherein the less transparent material comprises a metalloid. 566. The microparticle of claim 548, wherein the less transparent material comprises aluminum.

567. The microparticle of claim 548, wherein the less transparent material comprises tungsten, chromium, titanium, tantalum or molybdenum.

568. The microparticle of claim 548, wherein the less transparent material comprises silicon or aluminum, and the more transparent material comprises silicon dioxide, 569. The microparticle of claim 548, having a six sided shape with four elongated sides and two end sides.

570. The microparticle of claim S69, wherein the code of the encoded microparticlo is detectable regardless of which of the four elongated sides the barcode is disposed on.

571. The microparticle of claim 548, further comprising a biological material immobilized on a surface of the more transparent material. 572. The microparticle of claim 543, wherein the biological material comprises a segment of DNA,

573. The microparticle of claim 548, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

574. The microparticle of claim 573, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

575. The microparticle of claim 573, wherein, a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

576. The microparticle of claim 548, wherein &om2 to 15 portions of less transparent material are provided within the encoded microparticle. 577. The microparticle of claim 545, wherein flora 3 to 10 portions of less transparent material are provided within the encoded microparticle.

578. The microparticle of claim 545, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle.

579. The microparticle of claim 545, wherein the portions of less transparent material have varying lengths, 580, The microparticle of claim 548, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

581. The microparticle of claim 548, wherein the eortions of more transparent material and the portions of less transparent material all have varying lengths.

582. The microparticle of claim 548, that has a size that undergoes Brownian motion when in a buffer. 583. The microparticle of claim 548, wherein the more transparent material is formed of a spin-on material.

584. The microparticle of claim 548, wherein the more transparent material is a siloxane or silsesquioxane material.

585. The microparticle of claim 543, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous, 588. The microparticle of claim 548, wherein the portions of less transparent material are fu lly surrounded by the more transparent material. 587. The microparticle of claim 548, wherein the less transparent material comprises polysilicon.

588. The imicropaitiole of chum 548, disposed within a buffer in a well of a multi-well plate.

589. The microparticle of claim 548, wherein the alternating portions of more and less transparent material form a non-binary code.

590. The microparticle of claim 548, wherein the alternating portions of more and less transparent material form S a binary code,

591. The microparticle of claim S48, wherein the biological material comprises a segment of RNA.

592. The microparticle of claim 591 , wherein the biological material is a DNA or BNA probe immobilized on a surface of the encoded πiicropartfcle at a density of from 10^z TOlO⁶Mm¹.

593. The microparticle of claim 591, wherein the DNA or KNA probe is bound to at least a portion of a 0 complementary biochemical material from a sample with a fluorescent tag.

594. The microparticle of claim 548, wherein the less transparent material comprises a magnetic material,

595. The rmcroparticle of claim 594, wherein the magnetic material comprises nickel, cobalt or iron.

596. The microparticle of claim 543, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portion; of the more transparent material therebetween. 5 597. The microparticle of claim 548, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon.

598. The microparticle of claim 597, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

599. The microparticle of claim 598, wherein the portions^' of the less transparent material reflect at least 50% of 0 the visible light incident thereon.

600. The micrσparticle of claim 597, wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon.

601. The microparticle of claim 548, wherein a ratio ofthe length to width is from 2:1 to 50:l.

602. The micioparticle of claim 601, wherein the ratio ofthe length to width is from 4: 1 to 20: 1. 5 603. The microparticle of claim 548, having a length of less than 50 um and a width of less than 10 um. 604, The microparticle of claim 603, having a length of less than 25 um and a width of less than 5 um, €05. The micropartieles of claim 603, having a length of less than 27 um and a width of less than 3 um.

606. The microparticle of claim 548, having a volume of from 5 to 500 um⁵.

607. The microparticle of claim 548, having a volume of from 10 to 200 urn³, 0 608. An encoded microparticle comprising an elongated body having an inner layer and an outer layer, wherein the outer layer comprises a luminescent material embedded within the outer layer.

609. The encoded microparticle of claim 608, wherein the first material is more transmissive to visible ax near- visible light than the second material.

610, The encoded microparticle of claim 609, wherein the more transparent material comprises silicon dioxide. 5 611. The encoded microparticle of claim 609, wherein the more transparent material is an organic material.

612. The encoded microparticle of claim 609, wherein the more transparent material folly encapsulates the portions ofthe less transparent material.

613. The encoded microparticle of claim 609, wherein the more transparent material comprises silicon nitride,

614. The encoded ∞iciroparticle of claim 609, wherein the detectable code is detectable under reflected light 0 from the microparticle,

615. The encoded micropaiticfe of claim 609, wherein the detectable code is detectable under transmitted light passing through the microparticle.

616. The encoded microparticle of claim 609, wherein the less transparent material comprises silicon.

617. "Die encoded microparticle of claim 609, wherein the leas transparent material comprises amorphous silicon.

618. The encoded microparticle of claim 609, having a six sided shape with four elongated sides and two end 5 βides.

619. The encoded micropaiticle of claim 618, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

620. The encoded microparticle of claim 6^"09, further comprising a biological material immobilized on a surface of the more transparent material. 0 621. The encoded microparticle of claim 620, wherein the biological material comprises at least a portion of a nucleoside.

622. The encoded microparticle of claim 609, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions,

623. The encoded microparticle of claim 622, wherein the intermediate portions of more transparent material S have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

624. The encoded microparticle of claim 609, wherein a cross section taken orthogonal to the length of the encoded πricroparticle is substantially square.

625. The encoded microparticle of claim 609, wherein from 2 to 1 S portions of less transparent material are 0 provided within the encoded microparticle.

626. The encoded microparticle of claim 615, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle.

627. The encoded microparticle of claim 615, wherein the portions of less transparent material have varying lengths. 5 628. The encoded microparticle of claim 609, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

629. The encoded microparticle of claim 609, that has a size that undergoes Browiu^'aπ motion when in a buffer.

630. The encoded microparticle of claim 609, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous. 0 631. The encoded microparticle of claim 609, wherein the less transparent material comprises polysϋicoπ.

632. The encoded microparticle of claim 609, wherein the alternating portions of more and less transparent material form a non-binary code,

633. The encoded miαoparticle of claim 609, wherein the alternating portions of mor* and less transparent material form a binary code. 5 634. The encoded microparticle of claim 621 , wherein the nucleoside is a DNA or RNA probe immobilized on a surface of the encoded microparticle at a density of from 10² toloVum*.

635. The encoded microparticle of claim 609, whexein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

636. The encoded wicroparticlc of claim 609, wherein the portions of the less transparent material are discrete 0 and separate segments disposed with adjacent portions of the more transparent material therebetween.

637. The encoded micToparticle of claim 609, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon. 638, The encoded micioparticle of claim 637, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

639. The encoded micropartiole of claim 609, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon.

5 640, The encoded micropartiole of claim 639, wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon.

641. The encoded rnicroparticle of claim 609, wherein a ratio of the length to width is from Z: 1 to 50: 1.

642. The encoded rnicroparticle of claim 609, having a length of less «fy«" SQ xan and a width of less than 10 tun.

643. The encoded microparticle of claim 609, having a volume of from 5 to 500 urn⁵. 0 644. An encoded microparticle comprising; a spatially defined code; an outer surface layer of silicon dioxide that comprises within the layer fluorescent molecules.

645. The encoded microparticle of claim 644, where the outer surface layer is Strober grown fluorescent layer.

646. The encoded microparticle of claim 644, wherein the first material is more transmiasive to visible or near- S visible light than the second material.

647. The encoded microparticle of claim 646, wherein the moτe transparent material comprises silicon dioxide.

648. The encoded microparticle of claim 646, -wherein the more transparent material is an organic material.

649. The encoded microparticle of claim 646, wherein the more transparent material fully encapsulates the portions of the less transparent material. 0 650. The encoded microparticle of claim 646, wherein the more transparent material comprises silicon nitride.

651. The encoded microparticle of claim 646, wherein the detectable code is detectable under reflected light from the microparticle.

652. The encoded microparticle of claim 646, wherein the detectable code is detectable under transmitted light passing through the microparticle, 5 653. The encoded microparticle of claim 646, wherein the less transparent material comprises silicon.

654. The encoded microparticle of claim 646, wherein the less transparent material comprises amorphous silicon.

655. The encoded microparticle of claim 646, having a six sided shape with four elongated sides and two end sides. 0 656. The encoded microparticle of claim 655, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated sides the barcode is disposed on.

657. The encoded microparticle of claim 646, further comprising a biological material immobilized on a surface of the more transparent material,

658. The encoded microparticle of claim 657, wherein the biological material comprises at least a portion of a 5 nucleoside.

659. The encoded microparticle of claim 646, wherein each less transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions,

660. The encoded microparticle of claim 659, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the 0 intermediate portions are substantially the same regardless of location within the encoded microparticle.

661. The encoded microparticle of claim 646, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

662. The encoded microparticle of claim 646, wherein from 2 to 15 portions of less transparent material are provided within the encoded microparticle,

663, The encoded oπicroparticle of claim 662, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle. 664. The encoded microparticle of claim 662, wherein the portions of lees transparent material have varying lengths.

665. The encoded microparticle of claim 646, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths,

666. The encoded microparticle of claim 646, that has a size that undergoes Brownian motion when in a buffer. 667. The encoded microparticle of claim 646, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous,

668, The encoded mioroparticle of claim 646, wherein the less transparent material comprises polysilicon.

669. The encoded microparticle of claim 646, wherein the alternating portions of more and less transparent material form a non-binary code. 670. The encoded microparticle of claim 646, wherein the alternating portions of more and less transparent material form a binary code.

671. The encoded microparticle of claim 670, wherein the nucleoside is a DNΛ or RNA probe immobilized on a surface of the encoded microparticle at a density of δran 10* tol0*/um?.

672. The encoded microparticle of claim 671, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

673. The encoded microparticle of claim 646, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween.

674. The encoded microparticle of claim 646, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon. 675. The encoded microparticle of claim 674, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

676. The encoded microparticle of claim 646, wherein the portions of the less transparent material reflect at least 50% of the visible light incident thereon,

677. The encoded microparticle of claim 676, wherein the portions of the less transparent material reflect at leaet 80% of the visible light incident thereon.

678. The encoded microparticle of claim 646, wherein a ratio of the length to width is from2:l to 50:1.

679. The encoded microparticle of claim 646, having a length of less than 50 urn and a width of less than 10 urn.

680. The encoded microparticle of claim 646, having a volume of from 5 to 500 urn³,

681. An encoded microparticle comprising an elongated body having an inner layer and an outer layer, wherein the outer layer comprises a luminescent material bonded directly to the surface of the outer layer.

682. The encoded microparticle of claim 681 , where the outer βurface layer is Stiober grown fluorescent layer.

683. The encoded microparticle of claim 681, wherein the first material is more transmissive to visible or near- visible light than the second material.

654. The encoded microparticle of claim 681, wherein the more transparent material comprises silicon dioxide. 685. The encoded raicroparticle of claim 681, wherein the more transparent material is an organic material. 686. The encoded microparticle of claim 681, wherein the more transparent material fully encapsulates the portions of the less transparent material. 687, The encoded micropartJole of claim 681, wherein the more transparent material comprise,- silicon nitride. 68?. The encoded micioparticle of claim 681, wherein the detectable code Is detectable under reflected light from the microparticle.

689. The encoded microparticle of claim 681, wherein the detectable code is detectable under transmitted light passing through the microparticle.

690. The encoded microparticle of claim 681, wherein the less transparent material comprises silicon.

691. The encoded microparticle of claim 681, wherein the less transparent material comprises amorphous silicon.

692. The encoded microparticle of claim 681, having a six sided shape with four elongated sides and two end sides.

693. The encoded roicroparticle of claim 692, wherein the code of the encoded microparticle is detectable regardless of which of the four elongated aides the barcode is disposed on.

694. The encoded microparticle of claim 681, further comprising a biological material immobilized on a surface of the more transparent material. 695. The encoded microparticle of claim 694, wherein the biological material comprises at least a portion of a nucleoside.

696. The encoded microparticle of claim 681 , wherein each lesa transparent portion defines intermediate portions of more transparent material between adjacent two less transparent portions.

697. The encoded microparticle of claim 696, wherein the intermediate portions of more transparent material have lengths defined as the distance between two adjacent less transparent portions, and wherein the lengths of the intermediate portions are substantially the same regardless of location within the encoded microparticle.

698. The encoded microparticle of claim 681, wherein a cross section taken orthogonal to the length of the encoded microparticle is substantially square.

699. The encoded microparticle of claim 681, wherein £rom2 to IS portions of less transparent material axe provided within the encoded microparticle.

700. The encoded micropaiticle of claim 699, wherein from 4 to 8 portions of less transparent material are provided within the encoded microparticle.

701. The encoded microparticle of claim 699, wherein the portions of less transparent material have varying lengths. 702. The encoded micropaiticle of claim 683, wherein the potions of less transparent material have substantially the same lengths whereas the portions of more transparent material have varying lengths.

703. The encoded microparticle of claim 681, that has a size that undergoes Brownian motion when in a buffer.

704, The encoded microparticle of claim 681, wherein the more transparent material is a glass material comprising silicon, boron and/or phosphorous. 705. The encoded microparticle of claim 681, wherein the less transparent material comprises polysilicon.

706. The encoded microparticle of claim 681 , wherein the alternating portions of more and less transparent material form a non-binary code.

707._' The encoded microparticle of claim 681, wherein the alternating portions of more and less transparent material form a binary code. 708. The encoded microparticle of claim 707, wherein the nucleoside is a DNA or RNA probe immobilized on a surface of the encoded microparticle at a density of from 10² tolθ^β/urn².

709. The encoded microparticle of claim 708, wherein the DNA or RNA probe is bound to at least a portion of a complementary biochemical material from a sample with a fluorescent tag.

710. The encoded microparticle of claim 681, wherein the portions of the less transparent material are discrete and separate segments disposed with adjacent portions of the more transparent material therebetween. 711. The encoded raicroparticle of claim 681, wherein the portions of the less transparent material absorb at least 50% of the visible light incident thereon,

712. The encoded microparticle of claim 711, wherein the portions of the less transparent material absorb at least 80% of the visible light incident thereon.

713. The encoded microparticle of claim 681, wherein iht portions of the less transparent material reflect at least 50% of the visible light incident thereon.

714. The encoded microparticle of claim 693, wherein the portions of the less transparent material reflect at least 80% of the visible light incident thereon,

715. The encoded microparticle of claim 681, wherein a ratio of the length to width is from 2:1 to 50:1.

716. The encoded microparticle of claim 631, having a length of less than 50 um and a width of less than 10 urn. 717. The encoded microparticle of claim 681, having β volume of from 5 to 500 W,

718, An encoded microparticle comprising: a body with a plurality of surface indentations.

719, The microparticle of claim 718, comprising: a fluorescent material on a surface of the uαicioparticle.

720, The microparticle of claim 718, comprising; a fluorescent material embedded within a surface of the microparticle. 721. The microparticle of claim 718, wherein the fluorescent material forms a continuous film on said surface.

722. A method of fabricating micropartioles comprising: forming a layer on a substrate that has a plurality of indentations in the layer; planarizing said layer in order to control a depth of said indentations; patterning said layer to define individual microparticles; and releasing said microparticles in order to separate the microparticles from the substrate.

723. An encoded microparticle comprising: a body with an outer shell that comprises a fluorescent material therein.

724. A method of fabricating microparticles comprising: forming a layer on a substrate that has a fluorescent material therein; patterning said layer to define individual microparticles; and releasing said microparticles in order to separate the microparticles from the substrate.

725. A method for forming a raicroparticle, comprising: printing at a first time a first pattern for a microparticle; and printing at a second time after the first time a second pattern for the microparticle, wherein the second pattern is substantially orthogonal to the first pattern.

72$, A method for forming a microparticle, comprising: exposing at a first time a first pattern on a photoresist-, exposing at a second time a second pattern on the photoresist; and wherein the first and second patterns are substantially orthogonal and define at leas; in part a single microparticle.

727. A set of microparticles representing at least 100 different codes, wherein the micropartioles are elongated microparticles with spaced apart coding elements in a row, wherein the coding elements have variable lengths and are spaced apart with spaces therebetween each having substantially the same space length.

728. The set of microparticles of claim 727, wherein the different codes are detectable independent of the lengths of the spaces between coding elements.

729. The set of microparticles of claim 728, wherein the space length of the spaces is 1,5 microns or less.

730. The set of microparticles of claim 729, wherein the space length of the spaces is 1.0 microns or Jess.

731. The set of microparticles of claim 730, wherein the space length of the spaces is 0,5 microns or less.

732. A kit comprising a plurality of microparticles representing at least 100 different code., wherein the microparticles are elongated microparticles with spaced apart coding elements in a row, wherein the coding elements have variable lengths and are spaced apart with spaces therebetween each having substantially the same space length, and wherein different codes have different probes bound thereto.

733. A microparticle comprising an elongated body with coding elements in a row, wherein the coding elements have variable lengths and are spaced apart with spaces therebetween each having substantially the same space length and that is 1.5 microns or less.

734. The micrσparticle of claim 733, wherein the space length of the spaces is 1.0 microns oi less,

735. The microparticle of claim 734, wherein the space length of the spaces is 0,5 microns or less.

736. The microparticle of claim 733, wherein the body is comprised of glass.

737. The microparticle of claim 736, wherein the coding elements are opaque to light in the visible spectrum. 738. The microparticle of claim 737, wherein the coding elements reflect at least 50% of visible light incident thereon.

739. The πwcroparticle of claim 738, wherein the coding elements reflect at least 75% of the visible light incident thereon.

740. The microparticle of claim 739, comprising a probe attached to an outer surface thereof. 741. The microparticle of claim 740, wherein the total volume of the microparticle 1000 um^J,

742. The micxoparticle of claim 741_f wherein a maximum length of the microparticle is 50 ura.

743. The micropartiςlc of claim 741, wherein a maximum length of the microparticle is 25 urn.

744. The microparticle of claim 741, wherein a maximum length of the microparticle is 15 urn.

745. The microparticle of claim 733, wherein the coding elements are discrete elements rally separated by a light transmissive material.

746. The microparticle of claim 745, wherein the light transmissive material is glass,

747. The microparticle of claim 745, wherein each coding element is fully surrounded by glass on all sides.

748. The microparticle of claim 733, wherein the code of the microparticle is determined by a predetermined position of each space between coding elements. 749. The microparticle of claim 748, wherein the predetermined position is a center position.

750. A method for differentiating microparticles, comprising: providing a set of microparticles representing at least 100 different codes, wherein the microparticles are elongated microparticles with spaced apart coding elements in a row, wherein the coding elements have variable lengths and are spaced opart with spaces therebetween; determining a position but not a length of said spaces in each microparticle in order to determine a code defined by the coding elements in the row; and differentiating microparticles having different codes based on the determined positions of the spaces for each microparticle.

751. The method of claim 750, VYheiein the spaces in each miciopaxticlc have substantially the same length.

752. The method of claim 750, wheiein a first encoded micropartiole Is different &om the second micropaiticle S in a location of the space, wherein the difference is smaller than the minimum length of the coding elements.

753. The method of claim 750, wherein the length of the spaces is substantially equal to or less than a minimum length of a coding element.

754. A coding scheme foi encoded microparticles comprising: a) providing code elements that combine to form a code on an individual miciopaiticle; 0 b) code elements spaced apart from each other with predetermined spaces therebetween; c) wherein the predetermined spaces are smaller than the length of the code elements.

755. Aplurality of microparticles, comprising: a layer of microparticles arranged on a surface, wherein the microparticles are disposed substantially in a monolayer with substantially no micioparticles disposed or extending outside the 5 monolayer; wherein the microparticles comprise spatial codes; wherein the microp&rticles comprise probes theieon; and wherein the microparticles are disposed on a first area of the surface and wherein the micioparticles in the monolayer cover more than 60% of the first area of the surface. 0 756, The plurality of microparticles of claim 755, wherein the microparticles in the monolayer cover more than 70% of the first area of the surface.

757. The plurality of microparticles of claim 756, wherein the microparticles in the monolayer cover more than 80% of the first area of the surface.

758. The plurality of microparticles of claim 755, wherein fewer than 10% of the microparticles are disposed or 5 extend outside the monolayer.

759. The plurality of microparticles of claim 755, wherein at least 70% of the total microparticles in the first area have codes that are detectable.

760. The plurality of microparticles of claim 759, wherein at least 80% of the total microparticlts in the first area have codes that are detectable. 0 761. The plurality of microparticles of claim 755, wherein the particles are disposed in the monolayer in a liquid,

762. The plurality of microparticles of claim 761, wherein the particles are disposed in an imaging buffer.

763. The plurality of microparticles of claim 758, wherein fewer than 5% αf the microparticles are disposed or extend outside the monolayer.

764. The plurality of microparticles of claim 763, wherein the particles in the monolayer can be identified with 5 an identification rate of at least 95%.

765. The plurality of micropartioles of claim 755 , wherein the microparticles are disposed in the monolayer at a density of at least 2,000 microparticles per square millimeter.

766. The plurality of microparticles of claim 765, wherein the microparticles ate disposed in the monolayer at a density of at least 5,000 microparticlcs pel square millimeter, 0 767. The plurality of microparticles of claim 755, where each microparticle is comprised of glass.

768. The plurality of microparticles of claim 755, where eaoh microparticle has a maximum dimension of 50 urn.

769. The plurality of miςropartiolβs of claim 755, farther comprising separate segments fully encapsulated by transparent material,

770. The plurality of microparticles of claim 755, further comprising biochemical moieties bound thereto.

771. The plurality of microparticles of claim 755, further comprising DNA bound theτeto. 772. The plurality of microparticles of claim 755, wherein the micTopsriicles are disposed in a buffer solution and undergo Brownian motion. 773. A plurality of microparticles, comprising: a layei of rnicroparticles arranged on a surface, wherein the microparticles are disposed substantially in a monolayer with substantially no microparticles disposed or extending outside the monolayer; wherein the microparticles comprise spatial codes; wherein the microparticles comprise probes thereon; and wherein the microparticles are disposed on a first area of the surface and are disposed in the monolayer at e density of at least 2,000 microparticleβ per square millimeter. 774. The plurality of microparticle of claim 773, wherein the microparticles are disposed in a monolayer at a density of at least 5000 microparticles per square millimeters. 775ι A method of detecting codes of microparticles, comprising: providing a set of microparticles, each microparticle comprising a spatial code extending in a line or plane; wherein a layer of the microparticles is arranged on an inner surface of a container during analysis, wherein the microparticles are disposed substantially in a monolayer on the inner surface; transmitting through or reflecting electromagnetic radiation from the microparticles and detecting the transmitted or reflected electromagnetic radiation in order to detect the spatial codes of the individual microparticles; wherein the microparticles are disposed on a first area of the inner surface of the container and are disposed substantially in a monolayer at a density of at least 2,000 microparticles per square millimeter.

776. The method of claim 775, further comprising: mixing the set of microparticles with a first test fluid to cause binding of probes on the microparticles with a corresponding βnalyte; washing the microparticles with a second wash fluid; and adding a third analyzing fluid in which the particles will be disposed during detection;

777. The method of claim 775, wherein at least 90% of the microparticles in the first area are disposed in the monolayer. 778. The method of claim 777, wherein at least 95% of the microparticles in the first area are disposed in the monolayer.

779. The method of claim 778, wherein at least 99% of the microparticlea in the first area are disposed in the monolayer,

780. The method of claim 775, wherein the microparticlea undergo Browαian motion during analysis of the microparticles in the container.

781. The method of claim 776, wherein the second and third fluids are the same.

782. The method of claim 776, wherein the first and second fluids are the same.

783. The method of claim 776, wherein the first, second and third fluids arc the same.

784. A method of detecting codes of microparticles, comprising: providing a set of microparticlea, each microparticle comprising a spatial code extending in a line or plane; 5 wherein a layer of the microparticles is arranged on an inner surface of a container during analysis, wherein the microparticles are disposed substantially in a monolayer on the inner surface; transmitting through or reflecting electromagnetic radiation from the microparticles and detecting the transmitted or reflected electromagnetic radiation in order to detect the spatial codes of the individual micropaiticles; 1 Q wherein the microparticles arc disposed on a first area of the surface and wherein the microparticlea in the monolayer cover more than 60% of the first area of the surface.

785. The microparticles of claim 755, wherein the particles are in an image buffer.

786. An imaging system comprising: a well comprising a plurality of microparticks and a liquid therein;

15 wherein the microparticles are elongated and area arranged in a two dimensional layer at a density of 2000 particles per square millimeter; a source of electromagnetic radiation; a detector disposed to detect electromagnetic radiation after it is incident on the microparticles.

787. An imaging system comprising:

20 a well comprising a plurality of micropaiticles and a liquid therein; wherein the microparticles are disposed on a first βrea of a surface of the well and wherein the microparticles in the monolayer cover more than 60% of the first area of the surface. a source of electromagnetic radiation; a detector disposed to detect electromagnetic radiation after it is incident on the microparticles. 25 788. A biochemically active encoded microparticle comprising an elongated member comprising a code detectable when illuminated with light in the visible spectrum, wherein the microparticle has a volume of from 5 to 500 urn³, and wherein the microparticle is disposed in a liquid and undergoes Brownian motion in the liquid. 789. The encoded microparticle of claim 788, wherein the code is detectable when light is directed on the microparticle and reflected to an image capture device.

}0 790. The encoded tmcropaπicle of claim 788, wherein the code is detectable when light is directed on me microparticle and transmitted through the microparticle to an image capture device,

791. The microparticle of claim 788, wherein a 2 dimensional diffusion coefficient of the microparticles is greater than IxIO"¹² crrtVs.

792. The microparticle of claim 791, wherein a 2 dimensional diffusion coefficient of the microparticlea is 55 greater than IxIO'' ' cmVs.

793. A biochemically active encoded microparticle comprising an elongated member comprising a spatial code detectable when illuminated with light in the visible spectrum, and wherein the microparticle is disposed in a liquid and undergoes Brownian motion in the liquid.

794. The encoded microparticle of claim 793, wherein the code is detectable when light is directed on the to microparticle and reflected to an image capture device.

795. The encoded microparticle of claim 793, wherein the code is detectable when light is direoied on the microparticle and transmitted through the micropartiole to an image capture device.

796. The micropaiticle of claim 793, wherein a 2 dimensional diffusion coefficient of the microparticles is greater than 1x10"¹² cm²/s,

797. The microparticlc of claim 796^", wherein a 2 dimensional diffusion coefficient of the microparticles is greater than 1x10"" cm^z/s. 798. The microparticle of claim 793, having a volume of from 5 to 500 urn³.

799. The microparticle of claim 793, wherein the particle is elongated and has a length at least twice a width and at least twice a height of the πiicroparticle,

800. A monolayer of biochemically active encoded rjπicropartides, each comprising an elongated member comprising a code detectable when illuminated with light in the visible spectrum, wherein the microparticles are dispoaed substantially in a monolayer in a liquid, and wherein a 2 dimensional diffusion coefficient of the microparu^'clfis is greater than IxIO'¹² cm²/s.

801. A method for detecting an analyte in a test fluid, comprising: providing a set of biochemically active microparticles, each microparticle comprising a spatial code extending in a line or plane; wherein a layer of the microparticles is arranged on an inner surface of a container during analysis, wherein the microparticles are disposed substantially in a monolayer on the inner surface; transmitting through or reflecting electromagnetic radiation from the microparticlea and detecting the transmitted or reflected electromagnetic radiation in order to detect the spatial codes of the individual microparticles; wherein the microparticles undergo Brownian motion during the detection of the spatial codes.

802. The method of claim, 801, wherein a 2 dimensional diffusion coefficient of the microparticles is greater than lxlθ^"iz emVs.

803. The method of claim 802, wherein a 2 dimensional diffusion coefficient of the microparticles is greater than IxIO^'11 cra²/s. 804. A method for detecting an analyte in a test fluid, comprising: providing a set of microparticles, each microparticle comprising a spatial code extending in a line or plane; wherein a layer of the microparticles is arranged on an inner surface of a container during analysis, wherein the microparticles are disposed substantially in a monolayer on the inner surface; transmitting through or reflecting electromagnetic radiation from the microparticles and detecting the transmitted or reflected electromagnetic radiation in order to detect the spatial codes of the individual microparticles; wherein a 2 dimensional diffusion coefficient of the microparticles is greater than IxIO^"12 cmVs.

805. The method of claim 804, further comprising: mixing the set of microparticles with a first test fluid; washing the microparticles with a second fluid; adding a third analyzing fluid in which the particles will be disposed during detection.

806. The method of claim 805, wherein at least two of the first, second and third fluids are the same.

807. A method for detecting the spatial codes of elongated microbarcodcs, comprising: providing the elongated microbarcodes with a biological material thereon; binding the biological material on at least some of the microbarcodes with corresponding biological analyte in a test sample; providing the elongated rαicrobarcodes in a test fluid; transmitting through or reflecting electromagnetic radiation from the microparticles and detecting the transmitted or reflected electromagnetic radiation in order to detect the spatial codes of the individual microparticles; and detecting fluorescence on at least some of the microparticles in order to determine the presence of specific corresponding analyte in the test sample; wherein the microparticles undergo Brownian motion in the test fluid during the detection of the spatial codes and during the detection of the fluorescence. 808. A container comprising: a liquid; a plurality of microbarcodes in the liquid, the znicrobarcodes having a biochemically active material on surface; wherein the microbarcodes have a spatial code detectable under visible light; wherein the microbarcodes undergo Brownian motion in the liquid. 809. The container of claim 808, wherein the biochemically active material is a bio-material bound to a fworescently tagged complement.

810. The container of claim 808, wherein the microbarcodes comprise a glass outer layer. SU. The container of claim 810, wherein the microbarcodes comprise reflective or absorptive code segments within the glass. S 12. The container of claim 808, wherein the volume of a single microbarcode is 1000 \un³ or less.

813. The container of claim 808, wherein the container is a multi-well plate.

814. The container of claim 813, wherein the multi-well plate is a 96-welI plate.

815. A biochemically active encoded microparticle comprising an elongated member comprising a code detectable when illuminated with tight in the visible spectrum, wherein the microparticle has a volume of from S to SOO um\ and wherein the microparticle is disposed in a liquid and undergoes Brownian motion in the liquid,

816. A biochemically active encoded microparticle comprising an elongated member comprising a spatial code detectable when illuminated with light in the visible spectrum, and wherein the microparticle is disposed in a liquid and undergoes Brownian motion in the liquid.

817. A set of particles, the set comprising: at least 200 wells, each .well comprising a group of particles with the same code, but with a code different from codes in particles in the other wells; wherein each well comprises at least 100,000 particles. ,

818. The set of particles of claim 817, wherein at least SOO wells are provided,

819. The set of particles of claim 818, wherein at least 500,000 particles are provided in each well. 820. The set of particles of claim 81 S, wherein at least 1000 wells are provided.

821. The set of particles of claim 819, wherein at least one million particles are provided in each well.

822. The set of particles of claim 819, at least 200 wells are provided in a plurality of 96- well plates.

823. A set of wafers having unreleased particles thereon, the set comprising: at least 200 different particle codes formed on the wafers in corresponding wafer areas, each wafer having a plurality of wafer areas with different particle codes formed in each area, and wherein each wafer area comprises at least 100,000 particles.

824. The set of wafers of claim 823, wherein at least 500 wafer areas, each with particles having unique codes as compared to other wafer areas, are provided.

825. The set of wafers of claim 824, wherein at least 500,000 particles are provided on the wafers.

826. The set of wafers of claim 824, wherein at least 1000 wafer areas are provided. 827, The set of wafers of claim 825, wherein at least one million particles are provided in βaoh wafer area. 828. Λ kit comprising: a container; a buffer in the container; a plurality of micioparticies in the buffer, wherein different spatial codes are provided within different groups of microparticles; wherein each group of microparticles sharing the same spatial code has on their surface the same oligonucleotide, but differing from oligonucleotides of other groups; and wherein the number of groups sharing the same spatial code within the group is at least 1,200 with each group having a spatial code different from the other groups. 829. The kit of claim 828, further comprising instructions for testing a sample to detect complementary material to the oligonucleotides on the microparticles of at least one group,

830. A method comprising: providing the set of microparticles of claim 817; immobilizing probes on the microparticles, with a different probe going on each group of micioparticies with the same code; mixing the microparticles together to form a combined pool of microparticles; and taking an aliquot of the pool and placing in a separate container.

831. The method of claim 830, further comprising: using the aliquot to test a sample for the presence or absence of particular moieties. 832. The method claim 831, further comprising: wherein the testing the sample comprises identifying the rriicropartjcles with an identification rate of 95% or more.

833. A method comprising: providing the set of wafers of claim 823; singulating the wafers into individual wafer areas; placing each wafer area into a well; etching the microparticles with an etchant In each well so as to release the microparticles.

834. The method of claim 833, further comprising: immobilizing probes on the microparticles, with a different probe going on each group of microparticles with the same code

835. The method of claim 833, further comprising: mixing the released microparticles together to form a combined pool of microparticles; and taking an aliquot of the pool and placing in a separate container.

836. An oiπni-dircctiσπαl encoded particle comprising; a) a 1" material comprising two or more discrete segments aligned along an axis; b) a 2^nd material surrounding the 1^R material such that the segments are detectable through the 2^nd material; c) wkewby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis.

837. An omni-directional encoded particle made by the method comprising: a) providing a substrate;

S b) forming the particle with a code defined along an axis such that the axis is parallel to the substrate; c) whereby the code is formed by the segments and the code is detectable from ell directions perpendicular to the axis.

838. A method of forming an encoded particle with α lithography process to define a code for identifying the particle comprising the steps of: 0 a) providing a reticle field that defines a single code element per particle region; b) printing a 1" code element with the reticle field; c) printing a successive code element with the reticle Held such that the 1" and successive code element are within the same particle region; d) executing c at least once; 5 e) whereby a code on the particle ΪB provided.

839. Omni-directional encoded particle - 1" material, 2^nd material, code formed by segments, code detectable from all directions. a) collection of a multiplicity of microparticles where the code is different for at least two particles in the collection 0 b) particle is produced by a method that comprises photolithographic printing of the code c) > where photolithographic printing comprises printing the entire code by repeated printing of a single code element d) 1" material is opaque or reflective e) > I ° material is silicon 5 i) > 1" material is a metal g) 2^nd materials is transparent h) > 2^nd material is silicon dioxide, silica, or glass including glass doped with impurities, or silicon nitride i) particle has a bar shape with an aspect ratio equal to or greater than 2:1 and has an approximately square cross section perpendicular to the longest axis 0 j) particle has length less than 50um k) particle hsa a width less than 5um

1) particle contains a material or materials in addition to material 1 and 2 m) particles contains a magnetic material n) code scheme that uses discretization less than the dimension pf the code element 5 o) code scheme that uses code elements of a fixed width p) particles thai have biomolecules attached to the surface q) bioassay system that comprises particles according to claim 817

840. Omni-directional encoded particle - code formed along axis parallel to substrate

841. Method of forming encoded particle - printing successive code element w reticle field 0 a) code elements are gaps between discrete segments that form the code b) code elements are discrete segments that form the code c) code elements are regions that are optically distinguishable to provide the code d) code elements are holes, nothes or stripes e) code elements have a physical size less than lum (0.8um, O.Sum) f) particle has largest dimension less than 30um (50am, 70um) g) particle has an aspect ratio greater than 2: 1 h) particle has a width less than 8Um (SUm₉ SUm) i) particles are composed of one or more of the following materials: silicon, silicon dioxide, silicon nitride, glass, photoresist, aluπώauin, titanium, nickel, expoy, PMMA, polyimide, any polymer j) particles are composed of one or more material, deposited by chemical vapor deposition Ic) particle has a bar, rod, plate, or disk shape 1) code is present as a one or two dimensional pattern m) code is binary - has physical regions that have a one-to-one correspondence to the digits of a binary number n) code consists of the discrete locations of code elements where the discrete locations are βpecificed to a distance smaller than the code elements themselves o) printing is accomplished with a step-and-τepeat photolithograpic projection system (aka a stepper) p) printing is accomplished with a contact aligner photolithographic system q) printing exposes regions of a layer of positive photoresist, negative photoresist, or photo-curable epoxy

842, Coding scheme for encoded microparticles comprising code elements placed at discrete locations with me interval between the discrete locations being less than or equal to the physical dimension of the code elements. a) the interval is less than oτ equal to lum b) the interval is lees than or equal to 0.5um c) the size of the code elements is less than or equal to lum d) the particles are oπmi-diiectional encoded particles with each particle comprising a ."material comprising two or more discrete segments: aligned along an axis, and a 2^si material surrounding the I¹¹ material such that the segments are detectable through the 2^nd material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis e) the encoding features are produced with projection photolithography

843 , A multiplicity of biochemically active encoded microparticles that are arranged substantially in a monolayer on a surface, with the combined 2-dimensional area of the detectable particles occupying more than 30% of the surface area. Detectable particles refers to those that are able to be accurately identified.

844, An image of a multiplicity of biochemically active encoded microparticles that are arranged substantially in a monolayer on a surface, with the combined 2-dimensional area of the detectable particles occupying more than 30% of the total image area. the image is used to determine the result of a bioassay a) the panicles are omni-direcrlonal encoded particles with each particle comprising a I¹' material comprising two or more discrete segments aligned along an axis, and a 2^nd materia] surrounding the 1^st material such that the segments are detectable through the 2^βd material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis b) the encoding features arc produced with projection photolithography c) the particles are undergoing Brσwniaπ motion d) the code is comprised of code elements that have a physical size less than lum e) the biochemically active component is a nucleic acid, protein, antibody, enzyme, drug, receptor, or ligand f) the panicles have an elongated shape with an aspect ratio greater than 2; 1 g) the particles have a largest dimension of SOvuή

S h) the number density of detectable particles is greater than 2,000 particles per sqmm i) the combined 2-dimensional area of the detectable panicles ocoupys more than 50% of the total image area ,

845. A multiplicity of biochemically active encoded micrσparticles that are arranged substantially in a monolayer on a surface, with the number density of particles being greater than 2,000 particles per sqmm. 0 846. A system is provided that comprises: i) a multiplicity of encoded microparticles that contains a plurality of different codes, such that the tnicropartioles posβss biological moieties on their surface, and U) the multiplicity of microparticles are arranged in a monolayer on a 2D surface at a density higher than 2000 particles per sqmm, and iϋ) an apparatus is provided for conatining the particles in a carrier fluid, and iv) an imaging system is provided for the identification of the codes of the microparticles, and v) whereby a bioassay result is provided. S 847. A multiplicity of non-spherical encoded znicroparticles that are undergoing Brownian motion during image- based quantitation of a bioassay. a) the 2D diffusion coefficient of the particles is greater than Ix 1O'^U cmVs b) more than 10% of the particles are measured to undergo a lateral displacement of 20nm or greater in a time interval of one second or less 0 c) the particles exhibit fluorescence as a measure of molecular binding events d) the particles are omni-directional encoded particles with each particle comprising a 1*' material comprising two or more discrete segments aligned along an axis, and a 2""¹ material surrounding the 1" material such that the segments are detectable through the 2^nd material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis 5 e) the encoding features are produced with projection photolithography f) the particles are undergoing Brownian motion g) the code is comprised of code elements that have a physical size less than lum' h) the biochemically active component is a nucleic acid, protein, antibody, enzyme, drug, receptor, or ligand 0 i) the particles have a bar shape with an aspect ratio greater thaη 2: 1 & square cross section perpendicular to the longest axis j) the particles have a largest dimension of 50um

$48. A large set of biochemically active encoded particles that contains more than 200 codes and more than 100,000 particles per code. 5 a) the particles are omai-direetioπal encoded particles with each particle comprising a 1" material comprising two or more discrete segments aligned along an axis, and a 2^nd material surrounding the 1" material such that the segments are detectable through the 2^na material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis b) the encoding features are produced with projection photolithography 0 c) the particles are undergoing Brownian motion during quantitation of a bioassay d) the code is comprised of code elements that have a physical size less than lum e) the biochemically active component is a nucleic acid, protein, antibody, enzyme, drug, receptor, or ligand f) the particles have an elongated shape with en aspect ratio greater than 2: 1 g) the particles have a largest dimension o£50um

5 h) the large set of biochemically active encoded particles contains more than 1,000 cades i) the large set of biochemically active encoded particles contains more than 10,000 codes 849. Another preferred emodiment of the invention is a large set of biochemically active encoded panicles that: i) contains more than 1,000 codes, and ii) contains more than 100,000 particles per code, and iϋ) can be detected with an identification rate greater than 99%. Q 850. Encoded nticropartides with spatial encoding features less than lum in size a) the particle, are omni-directioπal encoded particles with each particle comprising a 1" material comprising two or more discrete segments aligned along an axis, and a 2^nd material surrounding the 1" material such that the segments are detectable through the 2ⁿⁱ material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis S b) the encoding features are produced with projection photolithography c) the position of the encoding features are positioned to within lOOnrα at 3 sigma accuracy d) the features are produced with projection photolithography e) the features are less than 600nm is size f) the particles are imaged and the encoding features are less than 5 pixels in size 0 8Sl. A multiplicity of non-spherical encoded particle, with a outer surface composed entirely of silica and a length less than 50um. a) the particles contain a spatially defined optical code b) the particles are omni-directional encoded particles with each particle comprising a 1" material comprising two or more discrete segments aligned along an axis, and a 2ⁿⁱ material surrounding the 1" 5 material such that the segments axe detectable through the 2^sd material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis c) the encoding features are produced witfi projection photolithography d) the particles are used in a bioasβay application e) the particles have a substantially square cross section 0 f) the particles are undergoing Brownian motion during quantitation of a bioassay g) the cods is comprised of code elements that have a physical size less than lum n) the biochemically active component is a nucleic acid, protein, antibody, enzyme, drug, receptor, or ligand i) the particles have an elongated shape with an aspect ratio greater than 2:1 5 j) the particles have a largest dimension of SOum

852, A computer program product that identifies the codes of encoded particles by associating discrete regions in an image into individual particles. a) the code is determined by extracting a pixel intensity profile from the axis of associated discrete regions 0 b) a bioassay result is computed as an output of the computer program c) the encoded particles consist of oroni-directional structures with opaque and transparent regions forming the code

853. An optical system used to image encoded microparticles that utilizes two CCD cameras for the simultaneous acquisition of a reflectance and fluorescence imago. a) used for detection to tøoassays

854. A flowcell provides the particles in a fluid for detection by continuous imaging. 85S. A spatially optically encoded miσropaiticle with a fluorescent outer layer. a) the particles are oπmi-directional encoded particles with each particle comprising a 1" material comprising two or more discrete segments aligned along an axis, and a 2^nd material surrounding the 1" material such that the segments are detectable through the 2^αd material, whereby a code is formed by the segments and the code is detectable from all directions perpendicular to the axis b) the encoding features are produced with projection photolithography

856. Square cross-section encoded microparticles are provided with increased interacting surface area for surface binding applications a) the particles have an elongated shape with an aspect ratio greater than 2: 1 b) where interaction of bioraolecules is determined by the presence of adjacent particles c) where interaction ofbiomolecules is determined by .the binding of the particles to a planar surface including a DNA, protein, or molecular microarray d) where interaction ofbiomolecules is determined by the binding of the particles to other reporter particles including fluorophores, quantum dots, latex or glass beads, colloidal metal particles, semiconductor nanorods, or any other encoded microparticles or nanopΦticfes. 857. An encoded πήcroparticle comprising: a) a 1" material comprising two or moie separate segments aligned along an axis; b) a 2^nd material surrounding the 1" material such that the segments are detectable through the 2^od material; c) wherein a code is formed by the segments. 858. An encoded particle xnade by the method comprising: a) providing a substrate with a planar surface; b) forming two or more segments aligned along an axis such that me is parallel to the planar surface of the substrate; c) releasing the particle from the substrate. $59, A method of producing an encoded particle comprising; a) providing a substrate with a planar surface; b) forming two or more segments aligned along an axis such that the is parallel to the planar surface of the substrate; c) releasing the particle from the substrate. S60. A method of producing an encoded particle comprising: a) providing a substrate; b) depositing a l" layer on the substrate; c) depositing a 2^nd layer on the substrate; d) forming the 2^nd layer into β plurality of particle regions such mat each particle region has two or more discrete segments aligned along an axis; e) depositing a 3rd layer OΛ the substrate; f) releasing the layers in the particle regions fiom the substrate to form individual particles; g) wherein a code is formed by the segments of an individual particle,

861. A method of producing an encoded particle comprising: a) providing a substrate with a planar surface; b) forming the particle with a code defined along an axis such that the axis is parallel to the planar surface of the substrate; o) wherein the code is detectable from all directions perpendicular to the substrate,

862, An encoded panicle made by the method comprising: a) providing a substrate with a planar surface; b) forming the particle with a code defined along an axis such that the axis is parallel to the planar surface of the substrate; c) wherein the code is detectable from all directions perpendicuar to the substrate. 863. An encoded micioparticle comprising: a) an elongated shape with an aspect ratio greater than 3:1; b) a square cross section along a plane perpendicular to the long axis of the particle. 864, An encoded micjoparticle comprising: a) a longest dimension less than 30um; b) an outer surface of silicon dioxide; c) a spatial code that is detectable in reflectance or transmission mode at a single collection wavelength.

865, A wafer substrate comprising: a) unreleased encoded microparticles on the surface of the substrate with each encoded microparticle having a code; b) more than X number of codes present; c) more than Y number of particles of each code, on average.

866, A wafer substrate comprising: a) unreleased encoded microparticles on the surface of the substrate with each encoded microparticle having a code; b) a density of microparticles greater than X microparticles per sqcrn.

867. A silicon wafer substrate having unreleased encoded microparticles comprising: a) a plurality of encoded microparticles such that the microparticles are bonded to the silicon substrate in a manner arising from the deposition of the constituent layers from which the microparticles arc formed; b) the microparticles having an entire outer surface composed of silicon dioxide (aka glass or silica).

868. A silicon wafer substrate having pre-release encoded microparticles comprising: a) a plurality of encoded microparticles such that the microparticles are bonded to the silicon substrate in a manner arising from the deposition of the constituent layers from which the microparticles are formed; b) the microparticles having an entire outer surface composed of silicon dioxide; c) the microparticles having the shape of a solid rectangular parallelepiped; d) the microparticles having β length and width measured parallel to the substrate surface and a height measured perpendicular to the substrate surface such that the width to height ratio is lee. than or equal to 5:1. 8ό9. A method of releasing encoded πiicroparticles from 8 silicon substrate without incorporation of a sacrificial layer comprising: a) bulk etching the silicon substrate; b) wherein released encoded πiicroparticles are provided.

S 870, A method of forming a plurality of encoded πucroparticles comprising: a) providing a planar substrate; b) forming a plurality of encoded microparticles on the surface of the substrate; c) releasing the encoded microparticles by bulk etching the substrate.

871 , A method of forming a plurality of encoded microparticles comprising: 0 a) providing a planaT substrate; b) depositing a sacrificial layer of silicon on the substrate; c) forming a plurality of encoded microparticles on the surface of the sacrificial layer; d) releasing the encoded microparticles by etching the sacrificial layer.

872. An encoded microparticle comprising: S a) en outer material of silicon dioxide; b) divits in one or more surfaces of the microparticle; c) the location of the divits forming the code on the microparticle; d) the divots not being detectable to read the code in reflection or transmission mode; «) the divots being detectable to read the code in fluorescence mode. 0 873. A method of forming a plurality of encoded microparticles on a planar substrate comprising the steps of; a) providing a substrate; b) depositing a code layer; c) forming topology in the code layer that exceeds S00A; d) depositing an overlying layer; 5 e) performing a planarization step on the overlying layer.

874. An encoded microparticle comprising; a) a spatially defined code; b) an outer surface later of silicon dioxide that contains fluorescent molecules; c) where the outer surface layer is grown by the Strober method. 0 875. A method of forming an encoded microparticle with a lithography process to define a code for identifying the particle comprising the steps of: a) providing a reticle field that defines a single code element per particle region; b) printing a 1" code element with the reticle field; c) printing a successive code element with the reticle field such that the 1 " and successive code element 5 are within the same particle region; d) executing (1) at least once more; e) wherein a code on the particle is provided,

576, A method of forming a plurality of encoded micrαparticles with a lithographic printing process to define codes for identifying the particles comprising forming the codes with more them one printing step. 0 877. A method of forming an encoded microparticle with a lithography process to define a code for identifying the particle comprising the steps of; a) providing a substrate comprising a plurality of encoded microparticles; b) providing a pattern comprising identification elements; c) printing the pattern on the substrate or a portion of the substrate; d) laterally shifting the substartc by a predefined distance; e) executing c at least once (more??);

S i) wherein a code on the particle is provided,

S78, A computer program directing a printing system to produce a set of encoded miαroparricles, the set comprising a plurality of regions, each region comprising a plurality of encoded rnicroparticles all having the same code, the computer program comprising a list of coordinate locations and lateral offsets that define the codes of the different regions. 0 879, A method of forming an encoded microparticle with a lithography process to define a code for identifying the particle comprising the steps of: a) providing a substrate comprising a plurality of encoded rnicroparticles; b) depositing a first layer; o) depositing a second layer; S d) forming particle regions according to stepper instructions; e) wherein a code on the particle is provided,

880. A method of forming a plurality of encoded microparticles with a photolithography process to define codes for identifying the particles comprising the steps of: a) providing a planar substrate on which the rnicroparticles are to be formed; 0 b) providing a 1 " reticle field having a 1" portion of an encoding pattern; c) exposing the substrate to the l" reticle field, thereby printing the 1 " portion of the encoding pattern; d) providing a 2™¹ reticle field having a 2^ad portion of an encoding pattern; e) exposing the substrate to the 2^nd reticle field, thereby printing the 2^nd portion of the encoding pattern; f) wherein a complete encoding pattern is provided with improved resolution, 5 Sδl, A coding scheme for encoded microparticles comprising: a) providing code elements that combine to form the code on an individual microparticle; b) code elements placed at discrete locations within a πύcroparticle; c) the discrete locations being smaller than the physical size of the code elements; d) whereby a high codespace coding scheme is provided. 0 882. A monolayer of a plurality of encoded rnicroparticles comprising: a) a container surface onto which microparticles are disposed; b) microparticles that have biochemical molecules attached to the surface of the microparticles; c) microparticles that have a spatially defined code; d) microparticles arranged in a monolayer on the container surface such that on average, (he area 3 occupied by the microparticles is greater than or equal to X% of the container surface area.

883, A monolayer of a plurality of encoded microparticles comprising: a) a container surface onto which microparticles are disposed; b) microparticles that have biochemical molecules attached to the surface of the microparticles; c) microparticles that have a spatially defined code; 0 d) microparticles arranged in a monolayer on the container surface such that on average, the number density occupied by the πύcropsrtjcfes is greater than or equal to X rnicropartioles per sqcm.

884. A multiplicity of spatially encoded microparticles that are undergoing Brownian motion during image- based quantitation of a bioasaay. 835. A system is provided that comprises; a) a multiplicity of encoded microparticles that contains a plurality of different codes, such that the S microparticles posess biological moieties on their surface; b) the multiplicity of microparticles are arranged in a monolayer on a 2D surface at a density higher than 2000 particles per sqmm; c) an apparatus is provided for conatining the particles in a carrier fluid; d) an imaging system is provided for the identification of the codes of toe microparticles; 0 e) whereby a bioassay result is provided.

886. A large set of biochemically active encoded particles that contains more than X codes and mare than Y particles per code on average.

887. An encoded microparticle comprising: a) a 1^st material comprising two or more discrete segments aligned along an axis; 5 b) a 2^nd material surrounding the 1 " material such that the segments are detectable through the 2^nd material; c) wherein a code is formed by the segments.

888. The encoded microparticle of claim 887, wherein the 1 " material is opaque.

889. The encoded microparticle of claim 887, wherein the l" material is a semiconductor including silicon, 0 single crystalline silicon, poly-crystalline silicon, amorphous silicon, doped silicon, germanium, SiCe, or GaAs.

890. The encoded microparticle of claim $37, wherein (he 1** material is a metal, including but not limited to aluminum, titanium, gold, silver, nickel, or cobalt.

891. The encoded microparticle of claim 887, wherein the 2^nd material is transparent.

892. The encoded micioparticle of claim 887, wherein the 2^nd material is silicon dioxide, silica, glass, doped 5 glass, or silicon nitride.

893. The encoded microparticle of claim 887, wherein the particle has the shape of a rectangular parallelpiped.

894. The encoded microparticle of claim 887, wherein the segments have the shapes of rectangular parallelpipeds.

895. The encoded microparticle of claim 893, wherein the particle has a square cross section 0 896. The encoded microparticle of claim 895, wherein the particle has a length less than or equal to 50unt

897. The encoded microparticle of claim 895, wherein the particle has a length less than or equal to 20um.

898. The encoded mioroparticle of claim 895, wherein the particle has a width less than or equal to 5 urn.

899. The encoded microparticle of claim 895, wherein the panicle has a width less than or equal to 2.5um.

900. The encoded microparticle of claim 895, wherein the particle has an aspect ratio equal or greater than 2:1. 5 901. The encoded microparticle of claim 895, wherein the particle has an aspect ratio equal or greater than 5:1.

902. The encoded microparticle of claim 887, wherein the I¹' material is composed of a multi-layer stack wherein one layer is a magnetic material.

903. The encoded microparticle of claim 887, wherein the 2^nd material is silicon dioxide that contains an outer layer of silicon dioxide impregnated with fluorescent molecules, 0 904. The encoded micioparticle of claim 887, wherein the 2ⁿⁱ material has biochemical molecules bound to the surface such as DNA, RNA, oligonucleotides, proteins, peptide, polypeptides, or small molecules. 905. An encoded microparticle comprising: a) a longest dimension measuring less than X um; b) a code that can be read in a flow cytometer with a single forward scatter detector.

906, A computer program product that identifies the codes of encoded panicles by associating discrete regions in an image into individual particles.

907. A binding assay comprising: a) substantially square cross section encoded microparticles; b) the presence of an analyte indicated by toe presence of particle-particle or particle-surface complexes.