US 20050012929 A1
A universal microplate analyzer capable of carrying out measurements on samples contained in the wells of microplates by fluorescence, absorbance, luminescence employs at least two light sources and optical fiber channels for directing excitation light to the sample wells. Flexibility of operation is provided by arrays of mirrors, apertures, and polarizers which can be positioned as required for the analysis to be carried out.
49. A universal microplate analyzer comprising:
(a) an illumination module including:
(1) as an excitation light source, a continuous wave light source and optionally, a flash light source;
(2) a means for shaping and filtering the light from said excitation light source;
(b) multiple optical fiber channels for transmitting the shaped and filtered excitation light to a read head or to the bottom of a microplate well;
(c) at least one read head including:
(1) a means for shaping and optionally polarizing the shaped and filtered excitation light received from a first one of said optical fiber channels;
(2) an optical switch including a mirror and beam splitter to reflect the shaped and optionally polarized excitation light to a sample in a microplate well;
(3) a means for shaping, filtering, and optionally polarizing light emitted by said sample in response to said excitation light directed into said sample; and
(4) a light detector for measuring the amount of said emitted light received from said means for shaping, filtering, and optionally polarizing light; and
(d) means for positioning a sample well by a plate carrier.
50. An analyzer of claim 1, further comprising a second one of said multiple optical fiber channels for transmitting shaped and filtered excitation light to the bottom of a microplate well and a third one of said multiple optical fiber channels for transmitting to said read head the light emitted by a sample in said microplate well from the bottom of said microplate well in response to said shaped and filtered excitation light.
51. An analyzer of
(c)(5) a means for shaping said emitted light received from said third optical fiber channel, and
(c)(6) a reflective mirror further included in said optical switch for directing the shaped emitted light received from said third optical fiber channel to said light detector.
52. An analyzer of
53. An analyzer of
54. An analyzer of
55. An analyzer of
56. An analyzer of
57. An analyzer of
The invention relates generally to equipment used to analyze chemical and biological samples in multiwell containers, commonly called microplates. Currently, they have up to 1536 wells in an array measuring about 3.4 by 5 inches (87×127 mm). Many types of analysis are possible, each one employing a light detector to determine the amount of light emitted from a sample in one of the wells. In general, analyses or measurements are made by fluorescence absorbance, or luminescence. Most analyses involve measuring the light emitted in response to excitation light directed into the sample or as the result of the introduction of chemical reagents. The present invention provides a means for carrying out each of these general types of analysis in a single instrument, a commercial embodiment of which is designated the Fusion™ Universal Microplate Analyzer by its manufacturer, the Packard Instrument Company.
Other analyzers which are able to carry out similar measurements on chemical and biological samples are disclosed in a large number of patents. Representative of more recent patents are the analyzers discussed in the following.
U.S. Pat. No. 6,042,785 assigned to Wallac OY shows an instrument capable of performing various spectrographic measurements. Two types of excitation light sources are used and two detectors for receiving light emitted from the sample, either passing through a mirror or through an aperture.
LJL Bio Systems in U.S. Pat. Nos. 6,097,028 and 6,071,748 shows a multi-functional analyzer which employs a plurality of excitation light sources and emitted light detectors. Optical switches are included to direct the excitation light and emission light to and from the sample. A feature of the instrument is its ability to limit the light to a “sensed volume” away from the walls of the sample container.
Lab Systems OY in U.S. Pat. Nos. 6,144,455 and 6,084,680 shows a fluorometer in which a partly reflective mirror has a plurality of areas transparent to excitation and emitted light and a plurality of areas which are non-transparent to excitation and emitted light.
The present inventors have sought to develop a multi-purpose analyzer capable of carrying out various types of measurements in an effective and efficient manner. Their universal microplate analyzer is described below.
In one aspect, the invention is a universal microplate analyzer, as shown in the accompanying illustrations and described below. The analyzer includes the following features:
In one aspect, the invention includes the universal microplate analyzer as configured for analyses by fluorescence, including time-resolved and polarization fluorescence, absorbance, and luminescence.
In another aspect, the analyzer of the invention also includes facilities for carrying out a Luminescent Oxygen Channeling Immunoassay (LOCI), such facilities being designated “Alpha Screen” by the Packard Instrument Company and described in U.S. patent application Ser. No. 09/512,707 and incorporated herein by reference.
Fluorescence, Absorbance, and Luminescence
The analyzer of the invention is used to determine the response of a sample (typically a liquid) to introduction of reagents which react with the sample to provide a measure of certain molecules in the sample. Such samples are often biological samples and when reagents are added, they may emit light with or without being activated by an external source of light (excitation light).
In general, excitation light will be limited to a certain range of wavelengths suitable for the sample being analyzed, while the sample will emit light having a spectral band characteristic of the sample which differs from the light used to excite the response. In other instances, the sample will emit light without use of an excitation light, but in response to reagents which have been added. The emitted light is detected, typically by a photo multiplier tube (PMT), and the quantity of light is used to characterize the sample. In the multi-functional analyzer of the invention, several types of analysis are possible.
In the simplest type of analysis, a sample is contacted with reagents which cause the sample to emit light (Luminescence). No excitation light is required. The light passes through an appropriate optical system and then is measured by the detector.
In another type of analysis, the amount of excitation light supplied to sample well from the bottom passes through an optical system, is measured and compared with the amount of light emitted when a sample is present in the well to provide a measure of the light absorbed by the sample and well (Absorbance).
Fluorescence involves measurement of emitted light from a sample which has been excited by a source of polarized or non-polarized light. Usually, the wavelengths characteristic of the emitted light will be different from that of the excitation light. In one type of analysis (Polarization Fluorescence) polarizing filters are used with both the excitation and emitted light. It is possible to carry out related fluorescence measurements which indicate the mobility of molecules in the samples by the degree to which polarization of the light has been changed by the sample.
In addition to three principal types of analysis just described, facilities may be included to carry out Luminescent Oxygen Channeling Immunoassay (LOCI), such facilities being designated “Alpha Screen” by the Packard Instrument Company.
External Appearance of the Analyzer
Interior Arrangement of the Analyzer
Now, having viewed the instrument from the outside,
The light transmitted to the sample well in microplate 42 will enter from either the top or the bottom, depending on the type of analysis being carried out. The analyzer of the invention makes it possible to carry out several types of analysis, including fluorescence (including time-resolved and polarization fluorescence), absorbance and luminescence. For certain types of analysis, including fluorescence, the light is introduced into the read head 12 via optical fiber channel 34 a through the side, where it passes through one of three available apertures 44 (in 28) and then is collimated by lens set 46 and in polarization fluorescence analysis the light is also polarized through one of two available polarizers 48 (in slide 29), before being passed into the top of the sample well in microplate 42 through additional lens 49 and 51. In an alternative embodiment, a liquid crystal polarization rotator is employed in combination with a fixed polarizer at 48 so that the polarization of the light can be varied electronically. Since the excitation light is intended to extend to the full width of the sample well, apertures 44 are not located adjacent the end of the optical fiber channel 34 a, but spaced away from it. The lens set is located at a distance from the optical fiber channel greater than would place the focus point of the lens set at the end of the optical fiber channel. With this arrangement of the aperture and the lens set the light beam widens, rather than being confined to a small region of the sample.
Two types of reflecting mirrors are provided by the analyzer of the invention. In the first type, the excitation light is reflected from a dichroic mirror 22 a (one of the possible selections of the optical switch 22), into the desired sample well in microplate 42, where it excites a response which depends on the nature of the sample. The emitted light from the sample has a different characteristic spectral band than that of the excitation light and it passes through the dichroic mirror 22 a (that is, not reflected as was the excitation light) to the light detector 18, e.g. a photo multiplier tube. Before reaching the light detector 18, the emitted light passes through one of a set of filters 50 to limit the band width, a lens set 52, if required a polarizer plate 54 (one of two available), an aperture 56 (one selected from four available) and light pipe 59. The detector 18 determines the amount of light received and computation is made by electronic circuits (not shown) of the property of the sample which correlates with the light emitted.
In the second alternative, the light encounters a beam splitter 22 b (either a thin-film beam splitter or a partly silvered mirror) which has been moved into position by the optical switch 22. The beam splitter 22 b does not selectively pass light above a predetermined wavelength cutoff as does dichroic mirror 22 a, but passes all wavelengths. A portion of the light passes through the beam splitter 22 b, and is absorbed by the beam dump 58 positioned opposite the light entry point. The remaining light is reflected into the sample from the top of the well in microplate 42. As before, the emitted light passes through the beam splitter 22 b in part and is directed to the light detector 18 via filter 50, lens set 52, polarizer plate 56 (if required), aperture 56, and light pipe 59 as previously described. While the beam splitter is not as efficient in directing light as a dichroic mirror, it has the advantage of providing flexibility that the dichroic mirror does not. That is, the beam splitter is capable of handling a wide range of light wavelengths, while the dichroic mirrors are limited to reflecting or transmitting light only below or above a predetermined wavelength.
Two types of beam splitters may be used. One type, a thin-film beam splitter, is made in a similar manner to a dichroic mirror. However, instead of reflecting light below a certain wavelength and passing light above that wavelength, a fraction of all the light is reflected, while the remainder passes through the beam splitter. A second type of beam splitter is a partially silvered mirror which also reflects a portion of the light, depending on the fraction of the surface which has been silvered. In one embodiment of the present invention the beam splitter employs a single elliptically-shaped silvered area in the center of a rectangular piece of glass, thereby making it possible to direct a narrow beam of light into a sample well, while the emitted light passed through the annular clear area around the reflective spot.
In a third method of operation, light is transmitted through one of the optical fiber channels 34 c to the bottom of the sample well in microplate 42, where it passes into the sample and excites a response from the sample having a different wave length. The emitted light exits the well in sample plate 42 through the bottom and is transmitted via another optical fiber channel 60 to a side of the read head 12 opposite that through which excitation light was introduced. The emitted light is collimated by a lens set 62 and then is reflected upward into the light detector 18. In this method, a reflective mirror 22 c is placed in position by the optical switch to reflect all of the light emitted from the sample. The reflected light passes through the emission filter 50, the lens set 52, aperture set 56 and the light pipe 59, before reaching the light detector 18.
A fourth method of operation involves the introduction of light into a sample from the bottom of the well in microplate 42 via optical fiber channel 34 b. The objective is to determine the amount of light absorbed, rather than the amount of emitted light. In this mode, all of the means used in the other three methods, i.e. the reflective mirror 22 c, the dichroic mirror 22 a, and the beam splitter 22 b are moved out of the light path by the optical switch 22 so that the light which passes through the sample 42 is directed upwardly into the light detector 18 following the path previously described. A diffuser is used with filter 50 to remove polarizing effects of the sample well, since the excitation light entering the sample well is randomly polarized by the transparent bottom of the well and varying from well to well. To avoid affecting the measurement made by the detector 18 the diffuser is used in this mode of analysis.
It is also possible to use the analyzer of the invention to make measurements of the luminescence of samples, that is samples which do not require light excitation. In such instances, when reagents are mixed with the sample (e.g. via injectors located in slots 53 in the lens just above the sample well), light is emitted by the sample. Light will be passed upwardly, usually without contacting any of the light diversion means described above (i.e., the optical switch 22), into the light detector 18 via filter 50, lens 52, aperture 56, and light pipe 59 for measurement of the amount of light emitted by the sample (polarizing filter 54 is not required). Alternatively, a dichroic mirror may be positioned in the emitted light path if desired since it will pass most of the light in the visible range.
As before, when light is directed into the top of a sample well either a dichroic mirror or a beam splitter is used to direct the excitation light and the emitted light. Both are capable of directing light, but they affect the light differently. A dichroic mirror is able to pass light above a predetermined wave length, but will reflect passage of light having shorter wave lengths. Thus, the excitation light is reflected into the sample well, while the emitted light, having a longer wave length band width than the excitation light, is able to pass through the dichroic mirror enroute to the detector. The term “beam splitter” could be interpreted as including a dichroic mirror also, but as used here, a beam splitter is either a partially silvered mirror or a thin-film beam splitter. In both types, light of the entire light spectrum is either passed or diverted. Part of the light is reflected and part passes through the beam splitter. Either type of beam splitter could be used. If a partially silvered mirror is used, it is preferred that a rectangular piece of glass having an oval-shaped silvered central spot is used to direct a narrow beam of excitation light into the sample well. When the emitted light from the sample well reaches the partially silvered mirror, the portion of the emitted light which is not reflected away from the central mirrored spot passes through the clear portion of the beam splitter to the photomultiplier tube.
While the analyzer of
In the analyzer of
In an analyzer having a liquid crystal polarization rotator-polarizer set, the excitation light can be changed electronically so that the polarization of the light is either parallel or perpendicular to the polarization of the emission polarizer.
In a preferred embodiment, the fixed polarizer is adjacent to a band-width filter placed in a multi-position wheel positioned in the path of the emitted light. Thus, the separate 2-position emission polarization plate 27 shown in
Description of the Analyzer in Each Mode of Analysis
Fluorescence measurements can be made in four modes:
The analyzer is programmed to place the proper optical elements in position for the selected analysis. In the first type of fluorescence a continuous incandescent lamp supplies excitation light, which is collimated by a first lens and then filtered to provide the desired wavelength range and finally the light is shaped by a second lens to provide an image of the light source to the selected fiber optic channel. The optic fiber channel terminates at one side of the read head, where the beam of light leaving the fiber optic channel is allowed to widen until it reaches an aperture which determines the size of the excitation light beam which will reach the sample well. It is a feature of the analyzer of the invention that, rather than confining the excitation light to a small region within the sample well, that the entire cross-section of the sample well receives the excitation light and emits light from the entire cross-section of the well. Following the aperture, a lens collimates the light and it passes to a partly-reflecting mirror, typically either a thin-film beam splitter or a partly silvered mirror, although use of a dichroic mirror is not excluded. The portion of the excitation light which is reflected 90 degrees toward the sample well then passes through two lenses before entering the sample well and the sample which had been placed therein. Since the partly-reflecting mirror passes only a portion of the excitation light to the sample well, the remainder of the light passes through the mirror and meets the beam dump, a feature of the invention. By absorbing the unused excitation light, the beam dump prevents the light from entering the the optical channel leading to the detector and affecting the measurement of the emitted light. The sample in the sample well emits light in response to the excitation light. The emitted light passes through the pair of lenses above the sample well and then through the partly-reflecting mirror and enters the portion of the read head containing optical elements associated with emitted light in all modes of analysis. First, the emitted light is passed through a filter to limit it to the band of light wavelengths to be measured. Then, the filtered light is passed through a lens to focus it, after which the light passes through an aperture to block stray light and enters the light pipe before reaching the detector.
In the second mode of analysis by fluorescence the excitation light from the continuous incandescent lamp is shaped and filtered as described above, but a separate optical fiber channel is used to direct the light to the bottom of the sample well. The light beam exiting the optical fiber channel widens to fill the cross-section of the sample well. Light emitted from the sample in response to the excitation light is received by the same optical fiber channel and transferred to the side of the read head located opposite to the entry port for the fiber optic channel discussed above which supplies excitation light to the top of the sample well. The emitted light passes through a lens to widen the light beam and then it is reflected by a fully-silvered mirror 90 degrees into the emitted light optics. The emitted light is passed through a band-pass filter to limit the light band width and then a lens narrows down (or focuses) the light and directs it to the light pipe as described above.
Time-resolved fluoresence uses substantially the same optical elements as those used in the first type of fluoresence described above, except that a flash lamp is used rather than a continuous incandescent lamp. Another difference is that, instead of a partly reflective mirror, a dichroic mirror is used, which is also partly reflective, but which reflects the portion of the light band below a cutoff wavelength value and passes light above that cutoff wavelength. The emitted light from the top of the sample well passes through the pair of lenses and through the dichroic mirror (that is, the portion of the emitted light having wavelengths above the cutoff value). The portion of the emitted light passing through the dichroic mirror then passes through the band-pass filter, the lens and aperture and finally through the light pipe before reaching the detector.
Polarization fluoresence is done using substantially the same optical elements as with the first type of fluoresence described above, except that both the excitation light and the emitted light are polarized as discussed above. The continuous incandescent lamp provides excitation light, which is shaped by a lens, filtered, and then passed through a second lens to provide an image of the lamp to the optical fiber channel. The light passes through the optical fiber channel and enters the read head, where it is allowed to widen and then passed through an aperture. The light is collimated by a lens and then passed through a polarizing filter. As explained above, polarizing filters are used to condition both the excitation and emitted light. During the analysis, the filters pass the light in the same direction at one time and at another time in directions perpendicular to each other. In one embodiment of the analyzer of the invention, filters having fixed orientation are switched into position as required to provide either parallel or perpendicular orientation of the light. In another embodiment, a liquid crystal polarization rotator is combined with a fixed polarizing filter having a simple orientation so that the light orientation can be changed electrically using the liquid crystal polarization rotator. The excitation light and emitted light paths are the same as described for the first type of fluoresence analysis, but, when polarization of the light is desired, the polarizing filters are inserted after the light has passed through a band-pass filter on the excitation path and band-pass filtered after polarization on the emission path.
Analysis by absorbance is carried out with the universal analyzer of the invention in a significantly different sequence of optical elements, but remaining within the general scope of the instrument already described. Either the continuous incandescent lamp or the flash lamp may be used. The excitation light is passed through a first lens, a band width filter and a second lens to form an image of the lamp at the optical fiber channel used for absorbance measurements. A mono-filament is used which terminates at the bottom of the sample well with a collimating lens, so that a very narrow light beam enters and leaves the sample well. The light enters through the bottom of the sample well, as was discussed in connection with one type of fluorescence above. However, the emitted light exits from the top of the sample well, then passes through the pair of lenses positioned immediately above the sample well and through an open space in the slide (optical switch) which contains the mirrors previously discussed, that is, the slide is moved so as to take the mirrors out of the way of the light. The light enters the emission optical section of the analyzer. In absorbence, a diffuser is also provided in order to remove the random polarization which has been found to be caused by the plastic bottom of the sample wells, which exhibit some pseudo-birefringence, that is not consistent among the sample wells. Since some detectors are sensitive to polarization of the light they receive, adding the diffuser improves the accuracy and consistency of the detector's measurements. After the diffuser, the light passes through a lens and is focused, then passes through an aperture and the light pipe to the detector.
Finally, luminescence measurements do not require many of the optical elements needed for the other types of analysis just described. No excitation light is used. Instead, reagents are introduced into the sample well and light emitted by the sample in response to the reaction which has been induced passes through the pair of lenses immediately above the sample well, through the open space in the mirror slide (optical switch) (as discussed in connection with absorbence), or alternatively the dichroic mirror may be used to pass the emitted light to the emission optical section. The emitted light passes through a band-pass filter, a lens to collimate the light, an aperture and finally the light pipe before reaching the detector.
Preferred Construction of the Principal Elements of the Universal Analyzer
Now that the operation of the analyzer has been discussed, the preferred construction of the principal elements will be described.
Supplemental Features of the Invention
In addition to the principal features of the universal analyzer of the invention, a number of other features may be included. These include: