US 20070148665 A1
An apparatus and method for imaging metallic nanoparticles is provided. Preferably, the invention provides for an apparatus and method for detection of gold colloid particles and for accurate reporting to the operator. The apparatus includes a substrate holder for holding the substrate, a processor and memory device, an imaging module, an illumination module, a power module, an input module, and an output module. The apparatus may have a stationary substrate holder and imaging module which are proximate to one another. The apparatus provided for a compact sized system without the need for complex motorized devices to move the camera across the substrate. Further, the apparatus and method provide for automatic detection of the spots/wells on the substrate, automatic quantification of the spots on the substrate, and automatic interpretation of the spots based on decision statistics.
51. In a substrate having a plurality of spots containing specific binding complements to one or more target analytes, one of the spots is a first test spot for metallic nanoparticles complexed thereto in the presence of at least a first target analyte, another one of the spots is a second test spot for metallic nanoparticles complexed thereto in the presence of at least a second target analyte, a method of automatically detecting at least some of the plurality of spots, the method comprising:
acquiring at least one image of the plurality of spots composed of metallic nanoparticles on a surface of the substrate;
compensating for at least one type of distortion in the at least one acquired image; and
automatically detecting at least some of the plurality of spots composed of metallic nanoparticles based on the at least one compensated acquired image.
52. The method of
53. The method of
54. The method of
55. The method of
56. The method of
wherein the at least one image is acquired using an image device; and
wherein acquiring the at least one image comprises acquiring the image without moving the image device and the substrate relative to one another.
57. The method of
58. The method of
59. The method of
wherein acquiring the at least one image comprises using an image device with a field of view; and
wherein correcting the grayscale distortion comprises applying a compensation model for brightness across the field of view of the image device.
60. The method of
61. The method of
62. The method of
generating a plurality of points distorted by the spatial distortion;
generating a plurality of points undistorted by spatial distortion;
generating a model based on the plurality of distorted and undistorted points; and
applying the model to the acquired image.
63. The method of
performing adaptive thresholding on at least a portion of the acquired image.
64. The method of
performing a threshold analysis on at least a portion of the acquired image so as to distinguish background areas of the acquired image from foreground areas of the acquired image; and
comparing characteristics of the foreground areas of the acquired image to characteristics of a predetermined spot area,
wherein the foreground areas having characteristics comparable to the characteristics of the predetermined spot area are deemed to be a spot.
65. The method of
wherein the characteristics of the foreground areas are selected from the group consisting of area, mass, shape, and circumference, and
wherein the characteristics of the predetermined spot area are selected from the group consisting of area, mass, shape, and circumference.
66. In a substrate having a plurality of spots containing specific binding complements to one or more target analytes, a method for automatically detecting at least some of the plurality of spots, the method comprising:
acquiring at least one image of the plurality of spots composed of metallic nanoparticles on a surface of the substrate;
compensating for at least one type of distortion in the at least one acquired image;
performing a threshold analysis on at least a portion of the acquired image so as to distinguish background areas of the acquired image from foreground areas of the acquired image;
comparing characteristics of the foreground areas of the acquired image to characteristics of a predetermined spot area, and
automatically detecting at least some of the plurality of spots composed of metallic nanoparticles based on the compensated acquired image,
wherein the foreground areas having characteristics comparable to the characteristics of the predetermined spot area are deemed to be a spot.
67. The method of
68. The method of
69. The method of
The current patent application claims priority to U.S. Patent Application Ser. No. 60/310,102 filed on Aug. 3, 2001 and entitled “Nanoparticle Imaging System and Method.” This application incorporates by reference U.S. Patent Application Ser. No. 60/310,102 in its entirety. The current patent application claims priority to U.S. Patent Application Ser. No. 60/366,732 filed on Mar. 22, 2002 and entitled “Method and System for Detecting Nanoparticles.” This application incorporates by reference U.S. Patent Application Ser. No. 60/366,732 in its entirety.
This present invention relates to detection of metallic nanoparticles. More specifically, the invention provides for methods and apparatuses for detection of gold colloid particles and for accurate reporting to the operator.
Sequence-selective DNA detection has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. DNA hybridization tests on oligonucleotide-modified substrates are commonly used to detect the presence of specific DNA sequences in solution. The developing promise of combinatorial DNA arrays for probing genetic information illustrates the importance of these heterogeneous sequence assays to future science.
Typically, the samples are placed on or in a substrate material that facilitates the hybridization test. These materials can be glass or polymer microscope slides or glass or polymer microtiter plates. In most assays, the hybridization of fluorophore-labeled targets to surface bound probes is monitored by fluorescence microscopy or densitometry. However, fluorescence detection is limited by the expense of the experimental equipment and by background emissions from most common substrates. In addition, the selectivity of labeled oligonucleotide targets for perfectly complementary probes over those with single base mismatches can be poor, limiting the use of surface hybridization tests for detection of single nucleotide polymorphisms. A detection scheme which improves upon the simplicity, sensitivity and selectivity of fluorescent methods could allow the full potential of combinatorial sequence analysis to be realized.
One such technique is the chip based DNA detection method that employs probes. A probe may use synthetic strands of DNA complementary to specific targets. Attached to the synthetic strands of DNA is a signal mechanism. If the signal is present (i.e., there is a presence of the signal mechanism), then the synthetic strand has bound to DNA in the sample so that one may conclude that the target DNA is in the sample. Likewise, the absence of the signal results (i.e., there is no presence of the signal mechanism) indicates that no target DNA is present in the sample. Thus, a system is needed to reliably detect the signal and accurately report the results.
One example of a signal mechanism is a gold nanoparticle probe with a relatively small diameter (10 to 40 nm), modified with oligonucleotides, to indicate the presence of a particular DNA sequence hybridized on a substrate in a three component sandwich assay format. See U.S. Pat. No. 6,361,944 entitled “Nanoparticles having oligonucleotides attached thereto and uses therefore,” herein incorporated by reference in its entirety; see also T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 289, 1757 (2000). The selectivity of these hybridized nanoparticle probes for complementary over mismatched DNA sequences was intrinsically higher than that of fluorophore-labeled probes due to the uniquely sharp dissociation (or “melting”) of the nanoparticles from the surface of the array. In addition, enlarging the array-bound nanoparticles by gold-promoted reduction of silver(I) permitted the arrays to be imaged in black-and-white by a flatbed scanner with greater sensitivity than typically observed by confocal fluorescent imaging of fluorescently labeled gene chips. The scanometric method was successfully applied to DNA mismatch identification.
However, current systems and methods suffer from several deficiencies in terms of complexity, reliably detecting the signal and accurately reporting the results. Prior art systems often times include large optics packages. For example, a typical imaging system may have a camera which is over 2½feet from the object plane (where the specimen sits). This large distance between the camera and the object plane results in a very large imaging device. Unfortunately, a large imaging system may occupy a significant portion of limited space within a laboratory. In order to meet this compact size requirement, other prior art imaging devices have reduced the distance between the camera and the object plane. While this reduces the size of the system, the small distance between the camera and the object plane can cause a great amount of distortion in the image acquired, with little distortion occurring at the center of the lens and with great distortion occurring around the outer portions of the image acquired. In order to avoid significant distortion and to increase the resolution in the acquired image, the camera is moved (or alternatively the substrate is moved) so that the center of the lens of the camera is at different portions of the substrate. Images are acquired at these different portions of the substrate and subsequently clipped at the images outer regions where the image is distorted. In order to reconstruct the entire image of the substrate, the clipped images are stitched together to form one composite image of the entire substrate. For example, a substrate may be divided into 100 different sections, with 100 images taken where either the camera or the substrate moves so that the center of the lens is centered on each of the 100 different sections. Each of the 100 images is then clipped to save only the image of the specific section. Thereafter, the entire image is reconstructed by pasting each of the 100 images together to form one composite image of the entire substrate. This type of prior art system is very complex in operation and design. Motors to move either the camera or the substrate are required, increasing cost and complexity. Further, because either the substrate or the camera is moving, the system is prone to alignment problems. Finally, because a series of images are taken, acquiring one composite image may take several minutes.
Further, imaging systems require an imaging module in combination with a personal computer. The personal computer includes a standard desktop personal computer device with a processor, memory, monitor, etc. The imaging module includes the camera, substrate holder, controller and memory. The personal computer sends control instructions to the controller of the imaging module and receives the images for processing. Unfortunately, this distributed system is expensive due to the additional cost of the personal computer and large due to the separate space required by personal computer.
Moreover, once the image of the substrate is acquired, there are several difficulties in terms of identifying spots or the wells on the substrate. “Well” is a term used to identify a separate test or experiment on or within the substrate. Each well might contain a different sample or a different test of the same sample. With regard to the spots, prior art systems may have difficulty distinguishing between the background of the substrate and the spots on the substrate. With regard to identifying wells, prior art systems and methods require the operator to identify the regions of the slide in order to identify the well that the imaging system will analyze. However, this requirement of operator input to identify the wells on a slide is inefficient and prone to error.
Further, current systems and methods are unable to detect small concentrations of nanoparticle probes which are under 50 nm (and in particular gold nanoparticle probes). Therefore, the prior art has been forced to use probes which are greater than 50 nm. However, these greater than 50 nm probes are more difficult to use from a processing standpoint. Alternatively, prior art methods have attempted to amplify the nanoparticle probes under 50 nm, such as by using silver particles, in order to compensate for being unable to detect the smaller nanoparticles. However, these attempts to amplify the nanoparticles have proven unworkable. For example, in the case of silver amplification, it has proven difficult to use because it is reactive with light and temperature (creating storage and packaging issues), is fairly expensive and is very difficult to reproduce results accurately. The prior art has thus frequently rejected the use of silver amplification.
Accordingly, the prior art solutions do not solve the problem of detecting nanoparticles in a practical manner.
The present invention relates to the detection of metallic nanoparticles on a substrate. The substrate may have a plurality of spots containing specific binding complements to one or more target analytes. One of the spots on the substrate may be a test spot (containing a test sample) for metallic nanoparticles complexed thereto in the presence of one or more target analytes. Another one of the spots may contain a control spot or second test spot. Depending on the type of testing at issue, a control or a second test spot may be used. For example, when testing for infectious diseases, a control spot may be used (and preferentially control positive and control negative spots) to compare with the test spot in order to detect the presence or absence of a nucleic acid sequence in the test sample. This nucleic acid sequence could be representative of a specific bacteria or virus. The control positive spot may be a metallic nanoparticle conjugated directly to the substrate via a nucleic capture strand, metallic nanoparticles printed directly on the substrate, or a positive result of metallic nanoparticles complexed to a known analyte placed in a separate well. A second test spot may be used when testing for genetic disposition (e.g., which gene sequence is present). For example, two test spots are used for comparison of gene sequences, such as single nucleotide polymorphisms.
In one aspect, an apparatus for detection of metallic nanoparticles, with or without chemical signal amplification of the metallic nanoparticles, is provided. The apparatus comprises a substrate holder for holding the substrate, a processor and memory device, an imaging module, an illumination module, a power module, an input module, and an output module. In one embodiment, the apparatus may have a stationary substrate holder and imaging module. This allows for imaging of a substrate by the imaging module without the need for motors to move either the substrate, the imaging module or both. Further, the apparatus may have an imaging module which is proximate to the substrate holder. In order to reduce the size of the imaging apparatus, the imaging module (such as a photosensor) is placed near the substrate holder (which holds the substrate). For example, the imaging module may be in the range of 30 mm to 356 mm from the substrate. Due to this close placement, the acquired image is subject to distortion, particularly at the edges of the acquired image. In order to process the acquired image better, the apparatus compensates for this distortion. For example, the apparatus compensates for grayscale distortion using a grayscale distortion model. As another example, the apparatus compensates for spatial distortion using a spatial distortion model. In this manner, the effect of the distortion in the acquired image is lessened.
In another aspect of the invention, a method for automatically detecting at least some of the spots on the substrate is provided. An image is acquired of the plurality of spots composed of metallic nanoparticles, with or without signal amplification, on the surface of the substrate. In one embodiment, the metallic nanoparticles are subject to chemical signal amplification (such as silver amplification). Alternatively, the metallic nanoparticles are not subject to chemical signal amplification. Optionally, an optimal image is obtained based on an iterative process. The obtained image is corrected for distortion, such as grayscale distortion and spatial distortion. The grayscale distortion correction may be based on a model that compensates for brightness degradation of the image. The spatial distortion correction may be based on a model that compensates for spatial deformation of the image. Based on the compensated image, at least a portion of the spots on the substrate are detecting in the acquired compensated image. Optionally, thresholding (and preferably adaptive thresholding) may be performed in order to distinguish the spots in the image.
In still another aspect of the invention, a method for automatically detecting at least one of the wells on the substrate is provided. The method includes the steps of automatically detecting at least a portion of the spots on the substrate and automatically determining the wells based on the automatic detection of at least a portion of the spots. The detected spots are analyzed to determine, from the unordered collection of detected spots, how the spots are organized into wells. One manner of analysis is to detect the spatial differences between the spots. Based on the spatial differences, the spots may be organized into wells. Moreover, patterns of the characteristics of the spots (such as characteristics due to differences in spacing) may be analyzed to detect how the spots are organized into wells.
In yet another aspect of the invention, a method for detecting the presence or absence of the one or more of the target analytes in the test spot on a substrate is provided. The substrate has a plurality of spots containing specific binding complements to one or more target analytes. One of the spots is a test spot for metallic nanoparticles, with or without signal amplification, complexed thereto in the presence of one or more target analytes. Another spot is a control spot or a second test spot for metallic nanoparticles complexed thereto in the presence of a second or more target analytes. The method comprises the steps of acquiring multiple images of the test spot and the control or second test spot, the multiple images being taken at different exposures and determining presence of said metallic nanoparticle complexes in the test spot as an indication of the presence of one or more of the target analytes based on the acquired multiple images of the spots. The multiple exposures may be taken based on an “optimal” exposure time for a portion of the image (preferably optimal for one well on the substrate) and an exposure time which is less than the optimal exposure time.
Thus, an advantage of the present invention to provide an imaging system within a compact housing.
Another advantage of the present invention to avoid the necessity of using complex motorized systems to move the camera across the substrate.
Still another advantage of the present invention is the ability to detect spots and/or wells on the substrate without expensive or complicated implementations.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, the appended claims and to the several views illustrated in the drawings.
The method and apparatus of the present invention relates to detection of metallic nanoparticles. In a preferred embodiment, the invention provides for methods and apparatuses for detection of gold colloid particles and for accurate reporting to the operator.
The examples set forth herein relate to an imaging system and method for detection of nanoparticles and in particular metallic nanoparticles. In a preferred embodiment, the nanoparticles are gold nanoparticles (either entirely composed of gold or at least a portion (such as the exterior shell) composed of gold) and amplified with silver or gold deposited post-hybridization on to the gold nanoparticles. The present invention may also be applied to other applications including, without limitation, detection of gold nanoparticles without silver or gold deposition.
As discussed in the background of the invention, there are several problems when detecting nanoparticles on a substrate including, for example: large sized systems occupying valuable space in a laboratory; complex motorized systems to move the camera across the substrate; problems in detecting spots and/or wells on the substrate that typically require expensive and complicated implementations. The present invention solves these and other problems of detecting nanoparticles in a manner that can be implemented for significantly less cost than current systems (less than US$10,000) and in an instrument footprint no larger than 18″ by 12″ by 12″.
“Analyte,” or “Target Analyte” as used herein, is the substance to be detected in the test sample using the present invention. The analyte can be any substance for which there exists a naturally occurring specific binding member (e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) or for which a specific binding member can be prepared, and the analyte can bind to one or more specific binding members in an assay. “Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof. The analyte can include a protein, a peptide, an amino acid, a carbohydrate, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.
“Capture probe” as used herein, is a specific binding member, capable of binding the analyte, which is directly or indirectly attached to a substrate. One example of a capture probe include oligonucleotides having a sequence that is complementary to at least a portion of a target nucleic acid and may include a spacer (e.g, a polyA tail) and a functional group to attach the oligonucleotide to the support. Another example of a capture probe includes an antibody bound to the support either through covalent attachment or by adsorption onto the support surface. Examples of capture probes are described for instance, in PCT/US01/10071 (Nanosphere, Inc.) which is incorporated by reference in its entirety.
“Specific binding member,” as used herein, is a member of a specific binding pair, i.e., two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule. In addition to antigen and antibody-specific binding pairs, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and captured nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence), complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, cells, viruses and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member. For example a derivative or fragment of the analyte, i.e., an analyte-analog, can be used so long as it has at least one epitope in common with the analyte. Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof including those formed by recombinant DNA methods or peptide synthesis.
“Test sample,” as used herein, means the sample containing the analyte to be detected and assayed using the present invention. The test sample can contain other components besides the analyte, can have the physical attributes of a liquid, or a solid, and can be of any size or volume, including for example, a moving stream of liquid. The test sample can contain any substances other than the analyte as long as the other substances do not interfere with the specific binding of the specific binding member or with the analyte. Examples of test samples include, but are not limited to: Serum, plasma, sputum, seminal fluid, urine, other body fluids, and environmental samples such as ground water or waste water, soil extracts, air and pesticide residues.
“Type of oligonucleotides” refers to a plurality of oligonucleotide molecules having the same sequence. A “type of” nanoparticles, conjugates, etc. having oligonucleotides attached thereto refers to a plurality of that item having the same type(s) of oligonucleotides attached to them.
“Nanoparticles having oligonucleotides attached thereto” are also sometimes referred to as “nanoparticle-oligonucleotide conjugates” or, in the case of the detection methods of the invention, “nanoparticle-oligonucleotide probes,” “nanoparticle probes,” “detection probes” or just “probes.” The oligonucleotides bound to the nanoparticles may have recognition properties, e.g., may be complementary to a target nucleic acid, or may be used as a tether or spacer and may be further bound to a specific binding pair member, e.g., receptor, against a particular target analyte, e.g, ligand. For examples of nanoparticle-based detection probes having a broad range of specific binding pair members to a target analyte is described in PCT/US01/10071 (Nanosphere, Inc.) which is incorporated by reference in its entirety.
Substrates and Nanoparticles
The method and apparatus of the present invention may detect metal nanoparticles amplified with a silver or gold enhancement solution from any substrate which allows observation of the detectable change. Suitable substrates include transparent or opaque solid surfaces (e.g., glass, quartz, plastics and other polymers TLC silica plates, filter paper, glass fiber filters, cellulose nitrate membranes, nylon membranes), and conducting solid surfaces (e.g., indium-tin-oxide (ITO, silicon dioxide (SiO2), silicon oxide (SiO), silicon nitride, etc.)). The substrate can be any shape or thickness, but generally will be flat and thin like a microscope slide or shaped into well chambers like a microtiter plate.
In practicing this invention, one or more different types of capture probes that bind to the target molecule are generally immobilized onto the surface of the substrate. The capture probe and the target molecule may be specific binding pairs such as antibody-antigen, receptor-ligand, and complementary nucleic acid molecules. See PCT/US)1/10071 (Nanosphere, Inc.) which is incorporated by reference in its entirety. The presence of any target molecule-capture probe complex bound to the substrate is then detected using nanoparticle probes. Methods of making the nanoparticles and the oligonucleotides and of attaching the oligonucleotides to the nanoparticles are described in PCT/US01/10071 (Nanosphere, Inc.) and PCT/US01/01190 (Nanosphere, Inc.), which are incorporated by reference in its entirety. The hybridization conditions are well known in the art and can be readily optimized for the particular system employed.
The capture probes may be bound to the substrate by any conventional means including one or more linkages between the capture probe and the surface or by adsorption. In one embodiment, oligonucleotide as capture probes are attached to the substrate. The oligonucleotides can be attached to the substrates as described in, e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039 (1996); Chrisey et al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucic et al., Chem. Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492 (1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591 (1992); and Hegner et al., FEBS Lett., 336, 452 (1993). A plurality of different types of capture probes may be arranged on the surface in discrete regions or spots in a form of an array which allows for the detection of multiple different target molecules or for different portions of the same target molecule.
The capture probes bound to the substrate surface specifically bind to its target molecule to form a complex. The target molecule may be a nucleic acid and the capture probe may be an oligonucleotide attached to the substrate having a sequence complementary to a first portion of the sequence of a nucleic acid to be detected. The nanoparticle-oligonucleotide conjugate has a sequence complementary to a second portion of the sequence of the nucleic acid. The nucleic acid is contacted with the substrate under conditions effective to allow hybridization of the oligonucleotides on the substrate with the nucleic acid or, alternatively, to allow hybridization of the nucleic acid with the nanoparticle-oligonucleotide conjugate. In yet another method the hybridization of the nucleic acid with the oligonucleotide on the substrate and the nucleic acid with the nanoparticle-oligonucleotide conjugate can be arranged to occur simultaneously. In one of these manners the nucleic acid becomes bound to the substrate. Any unbound nucleic acid and unbound nanoparticle-oligonucleotide conjugate is washed from the substrate before measuring the result of the DNA hybridization test.
The detectable change may be enhanced by silver staining. Silver staining can be employed with any type of nanoparticles that catalyze the reduction of silver. Preferred are nanoparticles made of noble metals (e.g., gold and silver). See Bassell, et al., J. Cell Biol., 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If the nanoparticles being employed for the detection of a nucleic acid do not catalyze the reduction of silver, then silver ions can be complexed to the nucleic acid to catalyze the reduction. See Braun et al., Nature, 391, 775 (1998). Also, silver stains are known which can react with the phosphate groups on nucleic acids.
Silver staining can be used to produce or enhance a detectable change in assays involving metallic nanoparticles performed on a substrate, including those described above. In particular, silver staining has been found to provide a huge increase in sensitivity for assays employing a single type of nanoparticle. For greater enhancement of the detectable change, one ore more layers of nanoparticles may be used, each layer treated with silver stain as described in PCT/US01/21846 (Northwestern University).
The oligonucleotides on the first type of nanoparticles may all have the same sequence or may have different sequences that hybridize with different portions of the nucleic acid to be detected. When oligonucleotides having different sequences are used, each nanoparticle may have all of the different oligonucleotides attached to it or, preferably, the different oligonucleotides are attached to different nanoparticles.
Alternatively, the first type of nanoparticle-oligonucleotide conjugates bound to the substrate is contacted with a second type of nanoparticles having oligonucleotides attached thereto. These oligonucleotides have a sequence complementary to at least a portion of the sequence(s) of the oligonucleotides attached to the first type of nanoparticles, and the contacting takes place under conditions effective to allow hybridization of the oligonucleotides on the first type of nanoparticles with those on the second type of nanoparticles. After the nanoparticles are bound, the substrate is preferably washed to remove any unbound nanoparticle-oligonucleotide conjugates. Silver stain treatment is then applied.
The combination of hybridizations followed by silver stain produces an enhanced detectable change. The detectable changes are the same as those described above, except that the multiple hybridizations result in a signal amplification of the detectable change. In particular, since each of the first type of nanoparticles has multiple oligonucleotides (having the same or different sequences) attached to it, each of the first type of nanoparticle-oligonucleotide conjugates can hybridize to a plurality of the second type of nanoparticle-oligonucleotide conjugates. Also, the first type of nanoparticle-oligonucleotide conjugates may be hybridized to more than one portion of the nucleic acid to be detected. The amplification provided by the multiple hybridizations may make the change detectable for the first time or may increase the magnitude of the detectable change. This amplification increases the sensitivity of the assay, allowing for detection of small amounts of nucleic acid.
If desired, additional layers of nanoparticles can be built up by successive additions of the first and second types of nanoparticle-oligonucleotide conjugates. In this way, the number of nanoparticles immobilized per molecule of target nucleic acid can be further increased with a corresponding increase in intensity of the signal.
Also, instead of using first and second types of nanoparticle-oligonucleotide conjugates designed to hybridize to each other directly, nanoparticles bearing oligonucleotides that would serve to bind the nanoparticles together as a consequence of hybridization with binding oligonucleotides could be used.
The Imaging System
The presently preferred embodiments of the invention will now be described by reference to the accompanying figures, wherein like elements are referred to by like numerals. Referring to
One type of sensor is a photosensor (not shown in
The imaging system also includes an illumination module 76. The illumination module 76 illuminates the sample with electromagnetic radiation. In one embodiment, the illumination module illuminates the sample with electromagnetic radiation in the visible light spectrum. Alternatively, light from other wavelengths such as infrared and ultraviolet may be used. Further, the illumination module may generate a specific wavelength of light, due to laser generation, or a spectrum of wavelengths, such as white light.
A variety of illumination modules may be used, such as side-lighting, front-lighting, and backlighting. Polarizers and filters can also be used to modify the incident light. When side-lighting, the illumination module may couple light to at least one side of the substrate so as to utilize the waveguiding capabilities of glass or another suitable substrate. Coupling of the illumination module to the support media may be accomplished in a variety of ways such as by a fiber optic bundle, a solid waveguide, a laser beam or LEDs glancing along the substrate.
The imaging module further includes at least one photosensor 74. A photosensor frequently used is a CCD or CMOS based sensor. The photosensor senses electromagnetic radiation, converts the sensed electromagnetic radiation into a data format and sends the data to processor 68. In a preferred embodiment, the sensor senses light in the visible light spectrum. Alternatively, the sensor may sense light in other bands of the electromagnetic spectrum, such as the infrared and ultraviolet bands. The photosensor, is composed of a plurality of pixels (e.g., 1.2 million pixels) although other size formats can be used. The amount of visible light which impinges on each pixel is converted into a data format. One such data format is a numerical value assigned to the amount of light which has impinged on the pixel. For example, if the data output of a pixel has a range of numerical values from 0 to 1023 (210 bits of data per pixel), 0 represents no light which has impinged on the pixel and 1023 represents saturation of the pixel. In this manner, if light impinges on the pixel after saturation, there is no change in the numerical value assigned. For example, the numerical value will remain at 1023 even if additional light impinges on the pixel after saturation. As discussed subsequently, the processor 68 may control the operation of the sensor (such as by controlling the exposure time) in order to modify the amount of light registered by the sensor.
Moreover, a lens or a series of lenses may be connected or coupled to the sensor to capture more of the scattered or reflected lightwave. In a preferred embodiment, the sensor works in conjunction with a single stationary lens, as shown in
The imaging system 50 may also include user input/output (I/O) 78. The user I/O 78 includes a display and a touch screen module as well as input scanners like a bar code wand. Alternatively or in addition, the user I/O may include a keyboard. The imaging module 50 further includes electronic I/O 80. The electronic I/O 80 may include data ports 55 which may interface with a network, such as a LAN, or may interface with an electronic device, such as a printer. The imaging system includes a power module, as shown at block 72. The power module 72 powers the various modules in the imaging system including the computer, the photosensor 74, the illumination module 76, the user I/O 78 and the electronic I/O 80.
The imaging system of
Further, the step of assigning the test and sample identification may be performed prior to or in parallel with the steps of spot detection, well identification and/or spot quantification. Alternatively, the step of assigning the test and sample identification may be performed after the steps of spot detection, well identification and/or spot quantification. As shown at block 100, the spots are quantified. This step of spot quantification is discussed in more detail in the flow chart of
As discussed above, one aspect of the invention is the automatic detection of the spots/wells on the support media. In a preferred embodiment, the software automatically detects the location of the wells in the image and identifies the locations of the specific areas in the well where spots of DNA have been deposited and hybridized. One method to the detection of wells is to use a series of image processing techniques to first extract some or all of the probable spots within the image. Then, analysis, such as geometric analysis, of the spot locations attempts to determine the location of the wells.
Detection of one, some or all of the spots on a substrate is difficult to perform. The surface area of the spots can be a very small portion of the entire image contributing to the difficulty of detecting the spots. For example, in the context of an image being composed of pixels, the spot may be on the order of 100 pixels or less within an entire pixel area of 1.2 million pixels. In addition, dirt, dust or the like may cause noise in the acquired image. Optionally, an “optimal” image of at least some (and preferably all) of the hybridized spots on the substrate is acquired. This “optimal” image may optionally be modified to correct for distortions in the image. After which, thresholding may be used to analyze the image to determine background (e.g., black portion of image) versus foreground objects (e.g., white portion of image). As one example of this background/foreground analysis, adaptive thresholding calculates the foreground/background separation based on a local neighborhood of image data values. The result typically is a collection of white areas against a black background. However, it can also be a foreground of dark spots against a relatively white background. The foreground areas of the image, derived by threshold analysis, may then be analyzed to determine whether these areas conform to a predetermined spot area. For example, characteristics of the foreground areas, such as area, mass, shape, circumference, etc. of the foreground areas, may be compared with predetermined characteristics of the spots, such as area, mass, shape, circumference, etc. If the characteristics of the foreground area(s) are comparable to the characteristics of the spots, the foreground area(s) is/are deemed a spot for purposes of well detection.
In the context of a sensor which measures light based on pixels, a pixel image (and preferably an “optimal” pixel image) is subjected to threshold analysis to separate foreground pixels from background pixels. After which, the image may be scanned to identify pixel clusters that define objects. These objects may then be arranged into “blobs” with these blobs being analyzed to determine their characteristics, such as area, mass, shape, circumference, etc. The characteristics of the blobs are then compared with the expected characteristics of the DNA spots to filter out noise. Referring to
There are several ways, discussed in more detail below, of modifying the amount of light registered to the sensor. In one aspect, the amount of light registered by the sensor may be controlled by the modifying parameters that control the operation of the sensor. Examples of parameters for the sensor include, but are not limited to, exposure time and sensor gain. In the case of exposure time, the amount of time for exposure of the sensor to the impinging light directly affects the amount of light registered by the sensor. Reducing/increasing the exposure time reduces/increases the amount of light. Where the sensor is a photosensor, the exposure time is modified by adjusting the time at which the pixels of the photosensor are read. Typically, for a digital sensor the exposure time controls an integration time. The sensor element values, the pixels, are read at the conclusion of the integration time. For example, if an exposure time of 60 mSec is desired, the photosensor is initialized and the pixel values are read 60 mSec after initialization. In another aspect, the amount of light registered by the sensor may be controlled by modifying parameters controlling the illumination module. Similar to the sensor, each illumination module has parameters controlling its operation. Examples of parameters for the illumination module include, but are not limited to, amount of time the illumination module is turned on, intensity of illumination module, etc.
Referring to blocks 106, 108 and 110, the flow chart iterates until an “optimal” image is obtained. An initial value of the exposure time for the sensor is chosen. Based on this initial exposure time, the photosensor reads the image, as shown at block 106. Because of noise in the system due to dirt, dust, etc., the image may optionally be despeckled, as shown at block 108. The despeckling may be achieved by applying a filter, such as a configurable median filter or mean filter in order to despeckle the image and remove any sharp signal spikes. A median filter considers each pixel in the image in turn and looks at its nearby neighbors to decide whether or not it is representative of its surroundings. The median filter replaces the pixel value with the median of neighboring pixel values. By contrast, the mean filter replaces the pixel value with the mean of neighboring pixel values. The median is calculated by first sorting all the pixel values from the surrounding neighborhood into numerical order and then replacing the pixel being considered with the middle pixel value. (If the neighborhood under consideration contains an even member of pixels, the average of the two middle pixel values is used.) After the image is despeckled, the pixels are read, as shown at block 110. Based on the read pixels, the processor 68 analyzes the pixels to determine whether the image is “optimal.” If the definition of “optimal” is based on the percentage of saturation of the pixels within the image, the processor 68 sums the amount of saturation within the image (e.g., determining the number of pixels within the image which are at saturation). If the percentage calculation is less than the “optimal” amount (i.e., less pixels are saturated than “optimal”), the exposure time is increased. Alternatively, if the percentage calculation is greater than the “optimal” amount (i.e., more pixels are saturated than “optimal”), the exposure time is reduced. The process iterates by changing the exposure time until the optimal image is obtained.
Once the optimal image is obtained, it is analyzed to determine the location of one, some or all of the spots on the substrate. To achieve an imaging system within a very small footprint for an instrument, the imaging system operates on a short optical working distance (i.e., the sensor is so close to the substrate). However, this short optical working distance results in an acquired image that is subject to distortion, particularly at the edges of the field of view. As discussed in the background of the invention, it is undesirable to limit the field of view since it would result in an undesirable requirement to move the camera across the object image, clipping and stitching, a series of images. Preferably and optionally, compensation of the acquired image is performed. Examples of distortion include, but are not limited to grayscale distortion and spatial distortion.
Distortion occurs when an optical system is forced to use a working distance that is shorter than desired for a given sensor size and field of view. This implementation is forced when marketing requirements force a low cost system, which mandates the use of off-the-shelf, high volume parts coupled with another market requirements, which is a small instrument footprint. In a preferred implementation, the low-cost, high-volume photosensor with a 9.7 mm horizontal is being forced to image a 65 mm horizontal field of view with a working distance somewhere between 30 mm and 356 mm. The resulting distortion grows as the working distance is reduced.
The distortion in the preferred embodiment manifests itself in both spatial deformation of the image and brightness degradation of the image. The distortion, both spatially and brightness, increases as a function of the distance from the center of the lens. An example of grayscale distortion is shown in
The data points accumulated with the 9 curves shown in
As shown in the data, the curves are a function of the brightness. The brighter the signal, the more pronounced the grayscale distortion effect on brightness. In one embodiment, one may gather a curve across the spectrum of brightness values (e.g., 65,536 for 216). In a preferred embodiment, by modeling 2nd order polynomial equations across the brightness spectrum, it can be determined that the 2nd and 1st order constants are linear and that the 0 order constants are related logarithmically.
The distortion caused by the lens also causes a spatial distortion between the image and the object. The distortion is severe enough that it does not permit reliable analysis of the image. In one aspect, the spatial distortion has a negative (barrel) distortion that compresses the edges of the image, as shown in
A data file that has the x and y translation necessary to move from the distorted point to the undistorted point is created from the image in
Given the eight known coordinates bounding each node the eight unknown coefficients can be found. In addition to calculating the corrected coordinates, one may interpolate the grayscale value since the corrected coordinates are not integral values. Since a digital image is discrete, non-integral coordinates do not exist. Simple solutions to this problem such as selecting the grayscale of the nearest integral neighbor introduce a number of undesirable artifacts into the resulting image. On the other hand, an optimal solution such as bicubic interpolation would introduce unacceptable computational requirements. Therefore, using estimation, another bilinear interpolation is performed using grayscale values of the four nearest neighbors as in the following relation:
After the acquired image is corrected for distortion, the shapes within the corrected image should be analyzed. Shape analysis operates on the binary images, in this case the foreground objects are white and the background is black. However, the inverse is also applicable under different illumination techniques. In one embodiment, a thresholding model is used to differentiate foreground and background objects in the grayscale image in order to generate a binary image suitable for shape detection and analysis. The thresholding model attempts to find a globally applicable separation between the foreground and background objects in order to generate a simple binary image suitable for shape detection and analysis.
However, since the substrate images often contain a non-uniform background and noise irregularities due to dust, scratches, etc, as well as irregularities in illumination, a preferred embodiment employs an adaptive thresholding algorithm, as shown at block 116. Adaptive thresholding calculates the foreground/background separation based on a local neighborhood of pixel values rather than attempting to find a globally applicable separation point based on histogram analysis.
Adaptive thresholding can be modeled in a variety of ways. One such method is by the following equations, considering that the ƒoriginal(x,y) is transformed into gbinary(x,y):
Once the foreground pixels have been separated from the background pixels using the adaptive thresholding model, one can scan the image and identify pixel clusters that define objects. Erosion and dilation is performed, as shown at block 118, in order to remove undesirable connections between foreground objects by separating blobs of pixel clusters connected together.
After pixel clusters have been detected and defined as single entities, blob detection builds data structures describing each cluster of connected foreground pixels as “blobs,” as shown at block 120. The blob detection algorithm traverses the pixel cluster data structures and builds two additional data structures and then computes blob metrics based on the new data structures.
The blob characteristics may then calculated, as shown at block 122. These objects (i.e., the portions of the images relating to the spots) are arranged into “blobs” that allow for spatial determination to filter out noise and blobs that do not have the expected characteristics of the DNA spots. Thus, different characteristics of the blobs may be calculated so that valid DNA spots may be accepted and invalid noise may be rejected. The different characteristics including without limitation: the blob's statistical shape moments; the blob's pixel area; the blob's pixel mass (sum of pixel values); the blob's centroid coordinates; the blob's circumference; and the blob's circularity coefficient.
The blob's statistical shape moments may be found by considering the shape of the blob to represent a function of two variables and then computing statistical moments. Moments are the basis of many of the subsequent blob metrics. Moments for a continuous function ƒ(x,y) are:
Another characteristic of the blob is the pixel area. The pixel area of a blob is the number of pixels in the blob. This is computed by counting the number of pixels represented by a blob's scan segment list. This value is the moment, m00.
Yet another characteristic is the blob's pixel mass (sum of pixel values). The pixel mass of a blob is the sum of the pixel values in the blob:
Another characteristic is the blob's centroid coordinates. A blob's coordinate location is computed by using moments about the x and y axis to determine a blob's average location:
Still another characteristic is the blob's circumference. A blob's circumference is computed by summing the distances between pixels in the blob's perimeter point list, represented by (xi, yi):
A final blob characteristic is the blob's circularity coefficient. Once the circumference and total area are known the circularity coefficient can be calculated:
Based on one, some or all of these blob characteristics, the blobs which register in the image may be analyzed and filtered to determine which are valid DNA spots and which are noise, as shown at block 124.
The spot detection steps provide the spots detected and the characteristics of the spots detected (such as area, circumference, etc.). Based on this, at least some of the detected spots are analyzed (and preferably geometrically analyzed) to determine, from the unordered collection of detected spots how the spots are organized into wells and rows.
Well identification takes the unordered collection of spots which have been detected (as discussed in the previous section) and attempts to automatically identify the spots which compose a well. This automatic identification does not require human operator intervention, as is required in prior art devices. Rather, the identification of the wells is based on attributes of the detected spots (such as spacing, patterns, etc.).
As discussed above, a substrate may be composed of a plurality of wells. Each of the wells may contain at least two spots (and preferably a plurality of spots). The spots within a certain well typically comprise one experiment so that the spots are related to testing for a particular target or series of targets. Well identification analyzes certain features of the detected spots, such as spacing between some or all of the detected spots, patterns for the detected spots, etc. in an attempt to obtain attributes about the well, such as the number of spots within the well, the location of the spots within the image acquired (e.g., in the case of pixels, which pixels groupings correspond with a particular spot), the geometry of the well, etc. A typical example of a well is a matrix of spots. The matrix may contain 3×3 spots (for a total of 9 spots in the well), 4×4 spots (for a total of 16 spots in the well), etc. depending on the particular substrate. For example,
The attributes in a well may be derived by analysis of the detected spots and/or by comparison of know characteristics of wells. In one aspect, the unordered spots are analyzed to determine the positive control spots within the wells. In a second aspect, dynamic measurement of spot to spot distances is used to differentiate spots within a well and differentiate spots within different wells.
Searching for an aspect of a well, such as an alignment row, follows the image analysis described above except that the aspects of the well deal with objects of a higher abstraction than the image processing. When the spots are defined in the image as detected “blobs,” the current set of all detected blobs may be filtered based on blob characteristics, such as blob area and blob circularity. Based on predetermined characteristics, the range of acceptable values of the blobs is configurable. This filtering removes blobs that are not likely to be valid hybridized spots. This is efficient and effective for subsequent processing that the data set is not too populated with extraneous objects that may randomly generate unintentional patterns.
Blobs that meet the predetermined filtering criteria for hybridized spots are collected into a new data set that represents the current set of probable hybridized spots, called the Total Spot Set. Once the Total Spot Set has been determined, an artificial image is constructed called the Indexed Intersection Image (I3). The software artificially renders the spot shapes into the I3 image using each spot's index value as the constituent pixel values. The I3 image allows the software to efficiently calculate intersection sets between spots and lines.
The forward row scan may begin at any aspect of the acquired image. In a preferred embodiment, the forward row scan begins at the top of the image and proceeds down toward the bottom of the image. The row scan first attempts to locate the alignment spot row for the upper row of wells and then attempts to locate the alignment spot row for lower rows of wells.
Once the upper alignment row has been properly located, the search for the lower alignment well may be aided by heuristic calculations that can be performed based on characteristics of the upper alignment row. The basic unit of computation in the forward row scan is the Spot Set. The Spot Set is initially defined by traversing a virtual line from the left edge to the right edge of the I3 image and collecting the intersected spots. The forward row scan moves forward by a specified number of pixel rows as long as the resulting spot set is empty. Once the initial spot set is non-empty, an iterative convergence may be performed to refine and raise the quality of the linear intersection of the spot set.
Several methods of spot set convergence may be used. Two example methods include a static method and a line-fit method. The line-fit method is able to tolerate a higher degree of variability in the input image. However, the line-fit method of convergence, by itself, may be unstable. The static convergence method does not tolerate a high degree of variability but it is very stable. Therefore, it is preferable to use static convergence of the spot set and then attempt to refine the spot set with the line-fit convergence. This combination produces an acceptable compromise between tolerance of variability and stability.
In static convergence, the software considers the spots to intersect a line with the equation y=mx+b but does not attempt to modify m, only b is modified. Additionally b is only modified such that it can increase, never decrease. In order to statically converge, the average y centroid of the current spot set is computed and then assigned a new b term as follows:
In line-fit convergence, the software considers the spots to intersect a line with the equation y=mx+b and attempts to adjust both m and b to properly converge the spot set. In order to perform a line-fit convergence, the software performs a least-squares line-fit on the centroid coordinates of the current spot set. The resulting line is used to define a new spot set. The process is iterated until two adjacent iterations produce identical spot sets. When attempting to line-fit converge, the software considers a configurable range of valid line slopes. The convergence is aborted if this slope range is exceeded. If the line-fit convergence is aborted the spot set produced by the static fit is chosen as a fallback and the processing continues as normal.
After the spot set has been refined and stabilized by the convergence iterations, a qualitative analysis of patterns present in the spot set is performed. To analyze the spot patterns, the software considers that the spot set is not unordered but rather represents spots intersecting along a line from left to right. As discussed above, known characteristics of the well may be analyzed to make conclusions regarding the unordered spots. Two characteristic elements of this linear spot pattern are the empty gaps between spots and the spot themselves. Analysis of the characteristic elements may take a variety of forms. One such form is to transform the spot set into an abstract symbolic form that facilitates symbolic pattern matching.
As shown at block 128, the spot and well gaps are computed. One of the fundamental elements of the spot set pattern is the gaps between spots along a line. The software may collect at least some (and preferably all) of the gap distances and attempts to group them into Gap Classes. A Gap Class is collection of distinct, measured, inter-spot gaps that are statistically similar such that they can be considered the same.
Based on the gaps computed, the number of spots within a well and/or the well pattern is determined, as shown at block 130. For example, based on the gaps computed, the layout of the particular well (number of spots, distribution of spots within well, layout, etc.) may be determined. To accomplish this, gaps are collected, coalesced using heuristics into classes, sorted, and then assigned symbols according to each gap class's frequency of occurrence along a line. The inter-spot gaps themselves are not assigned symbols, but rather each gap class is assigned a symbol. The symbols are represented by the letters a to ƒ.
The most frequently occurring gap class may be assigned a, the next most frequently occurring gap class is assigned, b, and so on. A number of heuristics may be used while assigning gap class symbols to protect against erroneous spot sets.
On a properly formed alignment row, the gaps between spots in a well's alignment row should be the most frequently occurring gap class, that is represented by the symbol a.
Once the gap class symbols have been assigned, they can be combined with the other fundamental element of the linear spot pattern, the spots themselves. Spots may be represented by the symbol S. Each actual inter-spot gap may be represented by the symbol corresponding to the gap class to which the actual gap belongs.
A linear spot pattern transformed into symbolic form may look similar to this example:
After the spot set has been transformed into symbolic form, the software may use a pattern matching mechanism based on regular expressions to determine if the current spot set represents a valid alignment row. The regular expression used to match an alignment row is configurable and contains definitions of subgroups that are used to delineate symbolic subsets that define each well.
During the building of the data structures representing spots, spot sets, gap classes, and the symbolic form of a linear spot set, the software maintains links between the various abstractions. These links enable backward traversal such that from the substring found by the regular expression matching, the software can determine the set of actual spots represented by the substring based on each symbol's string index.
Assuming the following regular expression:
For a valid alignment row, the software uses the links maintained between the abstractions to build a data structure representing spot clusters. Each spot cluster represents a group of spots horizontally along a line from left to right that make up the alignment row for one well. A detected well is defined by characteristics derivable from the well's spot cluster.
If the spot set is not a valid alignment row, then the software advances the current forward row scan past the current spot set and continues again to converge on another spot set. Advancing the current forward row scan past the current spot set is done by advancing the b term of the line equation without adjusting the m term. The b term is increased until two adjacent iterations produce different spot set.
Based on the determination of the number of spots in a well, a well mask is constructed, as shown at block 132. In the instance where an alignment row has been found, the software uses the metric data from actual spots in the alignment row to construct a mask of expected spots for each well. For example, where it is predetermined that the well's geometry is assumed to be square, there are as many spots down as there are spots across in the alignment row. In particular, if it is determined that the well is a 3×3 well based on pattern matching and if the alignment row (top three spots) have been found, the two lower rows may be found since the software knows that the two lower rows will line up, with three spots each, below the upper alignment row.
For each spot in an alignment row, a column of spot masks are interpolated underneath. When computing the vertical column of spot masks the software takes into consideration the linear equation representing the entire alignment row across the slide. The circular diameter of the interpolated spot masks is based on the average diameter of the alignment row spots of the entire slide.
Each interpolated spot's location is calculated as follows:
After the wells are identified, the individual spots within the wells are quantified. For example, the photosensor used to detect nanoparticles may saturate, limiting the amount of information which may be obtained from the image. An example of this problem is illustrated in
In order to extract usable information from the samples, the dynamic range of the sensors must be increased to allow for more useful information to be obtained in an area of interest within an image. This increase in the dynamic range is achieved by controlling the amount of electromagnetic radiation which is registered by the sensor. As discussed previously, in a preferred embodiment, controlling the amount of electromagnetic radiation registered by the sensor may be accomplished by modifying parameters which control the light incident on the sensor, such as exposure time, aperture size, etc. Moreover, other parameters which affect the amount of light registered on the sensor may be used. The data is then obtained based on the modified parameters of the sensor (e.g, different exposure times), as discussed subsequently in more detail. The data is subsequently analyzed in order to detect registration of nanoparticles, as discussed in the subsequent section.
Examples of data which may be obtained by modifying the amount of light registering on the sample are shown in
Alternatively, as shown in
As shown in
The image is then read, as shown at block 138. In this step, the portion of the image which is the current area of interest is read. For example, if well #1 was the first area of interest, the pixel values for well #1 (as determined in the well identification process) is read. As shown in
Then, it is determined whether an optimal exposure is sought, as shown at block 140. Before obtaining multiple exposures for a particular area of the substrate, it is preferred that an “optimal” exposure time be obtained. The “optimal” exposure time, as discussed above, may be defined as the amount of electromagnetic radiation registered by the sensor which, based on the sensor's characteristics, may best enable the detection of spots on the substrate. In the current example, the “optimal” exposure time may further be defined as being at or nearly at the outer boundary of the linear range of the sensor. In a preferred embodiment, the outer boundary of the linear range of the sensor may be quantified as a percentage saturation of the image. For example, the read pixels may be analyzed to determine if the optimal exposure time has been obtained, as shown at block 142. Specifically, the read pixels are analyzed to determine if a certain percentage (such as 1%) of the pixel vahles are at the saturation value. Based on the percentage determined, the exposure time is either increased (if less than the desired amount of pixels are saturated) or decreased (if more than the desired amount of pixels are saturated). After an optimal exposure time is found, the image may optionally be subject to correction due to grayscale and spatial distortion, as shown at blocks 144 and 146. These correction models were discussed above with respect to blocks 112 and 114 of
Since multiple exposures are sought in the linear range of the sensor, it is inquired whether additional exposures for a particular area (such as a well) are sought, as shown at block 150. For example, if four exposures are sought in the linear range and the “optimal” exposure was 100 mSec, three additional exposures are obtained for the area of interest at 25 mSec, 50m Sec, and 75 mSec. It is therefore preferable that the exposure times are evenly distributed within the range of 0 to the optimal exposure time. Alternatively, different exposure times may be selected within the range of 0 to the optimal exposure time. The system then iterates for the particular area of interest for the different exposure times. After all of the exposures are obtained for a certain area of interest, as shown at block 150, it is inquired whether there are any other areas of interest (i.e., any other wells to be analyzed), as shown at block 152. If there is another area, the program is repeated by first obtaining an optimal exposure for the area of interest, and then obtaining images at different exposure times.
Decision statistics analyzes the results of the spot quantification to determine conclusions.
Based on the output pixel values for the various spots, a “derived” pixel value may be determined by the regression analysis for a predetermined exposure time. In a preferred embodiment, the “predetermined” exposure time is chosen as the longest “optimal” exposure time. Other exposure times may be chosen for the predetermined exposure time. Based on this longest “optimal” exposure time, “derived” pixel values may be determined for each pixel within a well.
An example of the derived pixel values is shown in
The values shown in
In one embodiment, the intensity for an exposure time is determined based upon the function of the curve which is fit to data points in the second region 176. For example, to determine the intensity of the control sample, the value is extrapolated based upon the values in the second portion of the graph. This is shown by the dotted line in
The groups of spots in the well may be determined as target, positive control or negative control based on information supplied, such as test and sample identification, as shown at block 158. This step of determining the groups of spots may be performed before or after the regression analysis in block 154, the extrapolation/interpolation in block 156 and/or the calculations in block 160.
From these derived pixel values, a statistical analysis may be performed to determine whether the target spot is more like the control positive or control negative spot. Tests for infectious diseases where the outcome would be a positive result or negative result might use such an embodiment. Alternatively, the target spots can be compared directly to one another. Tests for genetic dispositions where the outcome is wild type, mutant or heterozygous might use direct comparisons of various target spots. The spots may be compared based on a summation of all of the derived pixels in a spot, an average value for the derived pixels in a spot, and the standard deviation for the derived pixels in a spot, as shown at block 160. From these values, statistical tests such as differences between means (t-Test, z-Test, etc.) may be performed, to compare spots and groups of spots, as shown at block 162. Alternatively the spots could be compared to each other with a percentage difference calculation or a ratio calculation.
Preferred embodiments of the present invention have been described herein. It is to be understood, of course, that changes and modifications may be made in the embodiments without departing from the true scope of the present invention, as defined by the appended claims. The present embodiment preferably includes logic to implement the described methods in software modules as a set of computer executable software instructions. A processor implements the logic that controls the operation of the at least one of the modules in the system, including the illumination module, the power module, the imaging module, and the input/output module. The processor executes software that can be programmed by those of skill in the art to provide the described functionality.
The software can be represented as a sequence of binary bits maintained on a computer readable medium described above, for example, as memory device 70 in
It should be understood that a hardware embodiment may take a variety of different forms. The hardware may be implemented as an integrated circuit with custom gate arrays or an application specific integrated circuit (“ASIC”). The embodiment may also be implemented with discrete hardware components and circuitry. In particular, it is understood that the logic structures and method steps described in the flow diagrams may be implemented in dedicated hardware such as an ASIC, or as program instructions carried out by a microprocessor or other computing device.
The claims should not be read as limited to the described order of elements unless stated to that effect. In addition, use of the term “means” in any claim is intended to invoke 35 U.S.C. §112, paragraph 6, and any claim without the word “means” is not so intended. Therefore, all embodiments that come within the scope and spirit of the following claims and equivalents thereto are claimed as the invention. This disclosure is intended to cover all variations, uses, or adaptations of the invention that generally follow the principles of the invention in the art to which it pertains.