United States Patent [19]
`He?'el?nger et al.
`
`US005784 1 5 2A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,784,152
`Jul. 21, 1998
`
`[54] TUNABLE EXCITATION AND/OR TUNABLE
`DETECTION MICROPLATE READER
`
`[75] Inventors: David M. He?'el?nger. San Pablo;
`Franklin R. Witney. Novato; Chris
`Cunanan. Bay Point. all of Calif.
`
`[73] Assignee: Bio-Rad Laboratories. Hercules. Calif.
`
`[21] Appl. No.1 729,111
`
`{22] Filed:
`
`Oct. 11, 1996
`
`Related US. Application Data
`
`[63] Continuation-impart of Ser. No. 405,468, Mar. 16, 1995,
`Pat. No. 5,591,981.
`
`[51] Int. Cl.‘5 ......................... .. GtllN 21/27; GOlN 21/64
`[52] us. 01. .......................... .. 356/73; 356/318; 356/417;
`356/436; 356/344; 250/4581
`[58] Field of Search ................................... .. 356/317. 318.
`356/417. 73. 436. 440. 344; 250/4581
`459.1. 461.1. 461.2
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3/1982 Hurni et al. .
`4,319,271
`2/1985 Banno et al. ......................... .. 356/440
`4,498,780
`4,626,684 12/1986 Landa .................................... .. 356/318
`4,786,170 11/1988 Groebler .
`4,877,966 10/1989 Tomei et al. .
`4,935,875
`6/1990 Shah et al. .
`4,968,148 11/1990 Chow et al. .......................... .. 356/436
`5,039,219
`8/1991 James et a1. .
`5,062,942 11/1991 Kambara.
`5,127,730
`7/1992 Brelje et a]. .
`5,138,170
`8/1992 Nogouchi .
`5,213,673
`5/1993 Fujimiya.
`5,290,419
`3/1994 Kambara .
`5.303.026
`4/1994 Strobl .................................... .. 356/318
`
`4/1994 Hiroshi eta]. 5,307,144 5,377,003 12/1994 Lewis et al. .
`
`
`5,381,016
`1/1995 Moriya.
`5,422,719
`6/1995 Goldstein.
`5,436,718
`7/1995 Fernandes et al. ................... .. 356/318
`
`FOREIGN PATENT DOCUMENTS
`
`68266 10/1991 Austria .
`2922788 12/1979 Germany .
`61-281945 12/1986 Japan .
`WO/90102l9 9/1990 WIPO .
`
`OTHER PUBLICATIONS
`
`Cothren. R.M; “Gastronomical Tissue Diagnosis by Laser
`-Induced Fluorescence Spectroscopy Endoscopy.” Gas
`trointestinal Endoscopy. vol. 36. No. 2 (Man/Apr. 1990). pp.
`105-111.
`Anderson. P.S.: “Auto?uorescenoe of Various Tissues and
`Human Skin Tumor Samples.” Lasers in Medical Science.
`vol. 12. No. 1. (Jan-Mar. 1987). pp. 41-49.
`Ried. T.; “Simultaneous Visualization of Seven Di?erent
`DNA Probes by in situ Hybridization Using Combinatorial
`Fluorescence and Digital Imaging Microscopy” Pmceed
`ings of the National Academy of Science of the United States
`of America. vol. 89. No. 4 (Feb. 15. 1992). pp. 1388-1392.
`
`(List continued on next page.)
`
`Primary Examiner—F. L. Evans
`Attorney, Agent, or Firm-David G. Beck; Townsend and
`Townsend and Crew
`
`[57]
`
`ABSTRACT
`
`A method and apparatus of analyzing samples contained in
`a microplate is provided. The instrument is capable of
`meastu‘ing ?uorescence. luminescence. and/or absorption
`within multiple locations within a sample well. The instru
`ment is tunable over the excitation and/or detection wave
`lengths. Neutral density ?lters are used to extend the sen
`sitivity range of the absorption measuring aspect of the
`instnlment. Due to the wavelength tuning capabilities of the
`instrument. the spectral dependence of the measured
`?uorescence. luminescence. and absorption of the materials
`in question can be analyzed. The combination of a data
`processor and a look-up table improve the ease of operation
`of the instrument. Several different formats are available for
`the output data including creation of a bit map of the sample.
`
`61 Claims, 6 Drawing Sheets
`
`Agilent Exhibit 1219
`Page 1 of 17
`
`

`

`5,784,152
`Page 2
`
`OTHER PUBLICATIONS
`Product Literature: Holographic Notch and Supem0tch®
`Filters. Kaiser Optical Systems. Inc. (1992).
`David M. Rust. “Etalon Filters." Optical Engineering. vol.
`33. No. 10. (Oct. 1994). pp. 3342-3348.
`Marc Solioz. “Video Imaging of Ethidium Bromide-Stained
`DNA Gels with Surface UV Illumination.” Biotechniques.
`vol. 16. No. 6 (1994). pp. 1130-1133.
`Product Literature: SDIOO & SDZOO Spectral Bio-Imaging
`Systems. Spectral Diagnosticslnc. (Nov. 9. 1994).
`Aaron T. Thompson. et al.. “Wavelength-Resolved Fluores
`cence Detection in Capillary Electrophoresis." Analytical
`Chemistry. vol. 67. No. 1 (Ian. 1. 1995). pp. 139-144.
`Product Literature: FMBIO 100 Fluorescent Imaging
`Device. Hitachi Software (1993).
`Product Literature: Model 373A DNA Sequencing System.
`Applied Biosystems.
`Leroy E. Hood et al.. “Automated DNA Sequencing and
`Analysis of the Human Genome.” Genomics 1. (1987). pp.
`201-212.
`
`K.B. Bechtol eta1.. “Using Dyes and Filters in a Fluorescent
`Imaging System.” American Biotechnology Laboratory
`(Dec. 1994). pp. 8-10.
`Product Literature: Digital Imaging Spectroscopy. Kairos.
`Inc.
`Christopher L. Stevenson et al.. “Synchronous Lumines
`cence: A New Detection Technique for Multiple Fluorescent
`Probes Used for DNA Sequencing.” Biotechniques. vol. 16.
`No. 6. pp. 104-1106.
`Product Literature: Tunable Filters. Cambridge Research
`Instrumentation. Inc.
`Product Literature: Biolumin Micro Assay System. Molecu
`lar Dynamics (1996).
`Product Literature: Victor's Technical Side, Continous Light
`Source For Flurometric And Photometgric Measurements.
`Wallac Oy. Finland & EG&G Berthold.
`Product Literature: Spectra Max 250 Microplate Spectro
`photometer. Molecular Devices (1994).
`
`Agilent Exhibit 1219
`Page 2 of 17
`
`

`

`US. Patent
`
`Jul. 21, 1998
`
`Sheet 1 of 6
`
`5,784,152
`
`,125 7
`
`FIG I
`
`Agilent Exhibit 1219
`Page 3 of 17
`
`

`

`US. Patent
`
`Jul. 21, 1993
`
`Sheet 2 of 6
`
`5,784,152
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`
`Agilent Exhibit 1219
`
`Page 4 of 17
`
`Agilent Exhibit 1219
`Page 4 of 17
`
`

`

`US. Patent
`
`Jul. 21, 1998
`
`Sheet 3 of 6
`
`5,784,152
`
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`Agilent Exhibit 1219
`Page 5 of 17
`
`

`

`US. Patent
`
`Jul. 21, 1998
`
`Sheet 4 0f 6
`
`5,784,152
`
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`Agilent Exhibit 1219
`Page 6 of 17
`
`

`

`U.S. Patent
`
`Jul. 21, 1998
`
`Sheet 5 of 6
`
`5,784,152
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`Agilent Exhibit 1219
`Page 7 of 17
`
`

`

`US. Patent
`
`Jul. 21, 1998
`
`Sheet 6 0f 6
`
`5,784,152
`
`FIG.‘ /2.
`
`Agilent Exhibit 1219
`Page 8 of 17
`
`

`

`1
`TUNABLE EXCITATION AND/OR TUNABLE
`DETECTION MICROPLATE READER
`
`5.784.152
`
`This is a Continuation-In-Part of U.S. patent application
`Ser. No. 08/405468. ?led Mar. 16. 1995. now US. Pat. No.
`5.591.981. the entire disclosure of which is incorporated
`herein by reference.
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates generally to rnicroplate
`readers and. more particularly. to a method and apparatus for
`measuring the luminescence. ?uorescence. and absorption
`of a sample in which the excitation and/or detection wave
`lengths are tunable.
`In the biotechnical ?eld. the ?uorescence and lumines
`cence properties of samples are routinely measured.
`Furthermore. it is often desirable to use a ?uorescent
`probe or dye to mark a particular biological structure such as
`a malignant tumor or a speci?c chromosome in a DNA
`sequence. and then use the ?uorescent probe or dye as a
`means of locating the structure. A variety of devices have
`been designed to read ?uorescent-labeled samples.
`In general. a device designed to read and/or image a
`?uorescent-labeled sample requires at least one light source
`emitting at one or more excitation wavelengths and means
`for detecting one or more ?uorescent wavelengths. Typically
`a device designed to read and/or image a luminescent
`sample requires means for detecting one or more wave
`lengths as well as means for adding one or more reagent
`lines. Reagents are typically added to the sample in order to
`initiate the luminescence phenomena. A device designed to
`measure sample absorption requires means for determining
`the amount of light transmitted through the sample in
`question. Furthermore. it is often desirable to determine the
`wavelength dependence of the transmittance.
`In US. Pat. No. 5.290.419. a multi-color ?uorescence
`analyzer is described which irradiates a sample with two or
`more excitation sources operating on a time-shared basis.
`Band pass ?lters. image splitting prisms. band cutotf ?lters.
`wavelength dispersion prisms and dichroic mirrors are use to
`selectively detect speci?c emission wavelengths.
`In US. Pat. No. 5.213.673. a multi-colored electrophore
`sis pattern reading apparatus is described which irradiates a
`sample with one or more light sources. The light sources can
`either be used individually or combined into a single source.
`Optical ?lters are used to separate the ?uorescence resulting
`from the irradiation of the sample into a plurality of ?uo
`rescence wavelengths.
`In US. Pat. No. 5.190.632. a multi-colored electrophore
`sis pattern reading apparatus is described in which one or
`more light sources are used to generate a mixture of light
`capable of exciting two or more ?uorescent substances. Both
`optical ?lters and di?’raction gratings are used to separate the
`?uorescence by wavelength.
`In US. Pat. No. 5.062.942. a fluorescence detection
`apparatus is described in which a ?uorescent light image is
`separated into a plurality of virtual images. Bandpass ?lters
`are used to separate the virtual images by wavelength.
`In an article by Cothren et al. entitled “Gastrointestinal
`Tissue Diagnosis by Laser-Induced Fluorescence Spectros
`copy at Endoscopy.” Gastrointestinal Endoscopy 36 (2)
`(1990) 105-111. the authors describe an endoscopic system
`which is used to study auto?uorescence from living tissue.
`The excitation source is monochromatic with a wavelength
`of 370 nanometers. Optical ?bers are used to collect the
`
`15
`
`25
`
`35
`
`45
`
`55
`
`2
`?uorescence emitted by the irradiated tissue. Emission spec
`tra are collected from 350 to 700 nanometers using an
`imaging spectrograph coupled to a gated optical multi
`channel analyzer. A sirnilar auto?uorescence system was
`described by Andersson et al. in “Auto?uorescence of Vari
`ous Rodent Tissues and Human Skin "llrmour Samples.”
`Lasers in Medical Science 2 (41) (1987) 41-49.
`Fluorescence analyzers in general su?er from a number of
`performance disadvantages. For example. typically such
`systems have a very limited selection of available excitation
`wavelengths; detection is generally limited to discrete wave
`length bands; the systems normally do not have the ability
`to measure luminescence and/or absorption; samples must
`be contained in one of only a few con?gurations; and if
`microplates are used. it is not possible to obtain multiple
`readings within a single sample well.
`From the foregoing. it is apparent that a microplate reader
`is desired which can measure the wavelength dependence of
`the ?uorescence. luminescence. and absorption properties of
`a sample at multiple locations within a single sample well of
`a microplate.
`
`SUMMARY OF THE INVENTION
`The present invention provides a microplate reader
`capable of making readings within multiple locations within
`each sample well of the rnicroplate. The apparatus measures
`?uorescence. luminescence. and absorption at each selected
`location. The excitation and/or detection wavelength is
`tunable. thus allowing the wavelength dependence of the
`various properties to be determined.
`The tuning section of the excitation and/or detection
`subassemblies can utilize dispersive elements. diffractive
`elements. ?lters. or interferometers. Examples of dispersive
`and di?'ractive elements are prisms and gratings. respec
`tively. Examples of ?ltas are short pass ?lters. long pass
`?lters. notch ?lters. variable ?lters. acousto-optic ?lters.
`polarization ?lters. interference ?lters based on continuously
`varying ?lm thickness. and tunable liquid crystal ?lters.
`Examples of interferometers include Fabrey-Perot etalons
`and common path interferometers.
`In one embodiment of the invention. the user inputs the
`type of sample container. i.e.. arnicroplate with 6. 12. 2A. 48.
`96. or 384 wells. The user can also select to analyze a gel or
`a storage phosphor plate. Once the type of sample and
`sample container are selected. the user enters the number of
`locations per sample well which are to be analyzed. The user
`can either select to use a predetermined test pattern or
`specify the actual testing locations. The user can also select
`to run a pseudo-continuous test pattern in which the micro
`plate is moved in a serpentine pattern during analysis. thus
`mapping out the entire microplate.
`A further understanding of the nature and advantages of
`the present invention may be realized by reference to the
`remaining portions of the speci?cation and the drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is an illustration of one embodiment of the
`invention;
`FIG. 2 is an illustration of one aspect of the optical train
`in one embodiment of the invention;
`FIG. 3 is an illustration of a Pellin-Broca prism;
`FIG. 4 is an illustration of a wavelength dispersive system
`using a grating;
`FIG. Sis an illustration of a dual ?lter wheel approach to
`obtaining wavelength tunability;
`
`Agilent Exhibit 1219
`Page 9 of 17
`
`

`

`5,784,152
`
`3
`FIG. 6 is an illustration of a SAGNAC interferometer;
`FIG. 7 is an illustration of a monolithic interferometer;
`FIG. 8 is an illustration of the cross-section of a multi
`well microplate containing 6 wells;
`FIG. 9 is a functional block diagram of an alternate
`embodiment in which the user can specify the actual loca
`tions within a sample well at which testing is to be per
`formed;
`FIG. 10 is an illustration of two scanning patterns for a 6
`well microplate;
`FIG. 11 shows a functional block diagram of one embodi
`ment of the detection system;
`FIG. 12 shows an alternate embodiment of the detection
`system; and
`FIG. 13 shows a third embodiment of the detection
`system.
`
`4
`operated either independently of or simultaneously with
`detector 111. Light from source 105 passing through sample
`101 is imaged onto detector 117 by imaging optics 119. Prior
`to being imaged. the transmitted radiation passes through a
`neutral density (hereafter. ND) ?lter 121. The value for the
`ND ?lter 121 is selected by the user (or by the system when
`operated in automatic mode). thus allowing detector 117 to
`measure a broad range of transmittances while operating in
`its optimal sensitivity range.
`Fixture 105 is coupled to a pair of positioners 123.
`Positioners 123 allow sample 101 to be moved in two
`orthogonal directions (i.e.. X and Y) with respect to source
`105. detector 111. and detector 117. Although in this
`embodiment sample 101 is moved. it is also possible to
`move the source and the detector(s) and keep the sample
`stationary. In this alternate embodiment. ?ber optics can be
`used to provide a ?exible optical light delivery and detection
`system.
`Although the system can be controlled manually. prefer
`ably a data processor 125 is used to control the various
`aspects of the system as well as store the output data from
`the detectors. In the preferred embodiment. processor 125 is
`coupled to tuning sections 107 and 115. ND ?lter system
`121. light source 105. focussing optics 109. detectors 111
`and 117. and positioners 123. Processor 125 also controls the
`temperature of incubator 102. Although processor 125 can
`be used to store the raw data from the detectors. preferably
`processor 125 places the data in a user de?ned format.
`Preferably processor 125 also controls the system gain
`settings. the sampling time. and the delay. if any. between
`the source ?ash and the sampling period.
`The present invention can be used for investigating both
`?uorescence and luminescence phenomena. Typically for
`?uorescence measurements. a probe is attached to the area
`of interest. for example a speci?c chromosome region.
`Currently the number of useful dyes is relatively limited. In
`order to increase the number of probes that may be imaged
`in a given experiment. combinatorial ?uorescence
`approaches have been developed. In a combinatorial
`approach ?uorescent reporter groups are used either singu
`larly or in combination. The table below illustrates how
`three ?uorescent reporters. A. B. and C can be used for up
`to seven probes. The number of detectable probes can be
`increased to ?fteen with four ?uorophores and to twenty six
`with ?ve dyes.
`
`Probe Number
`
`ReporterCombination
`
`55
`
`65
`
`Although a number of techniques for illuminating sample
`101 can be used with the present invention. FIG. 2 illustrates
`a speci?c con?guration which is well suited for investigating
`?uorescence phenomena. Light emitted from a source 201
`?rst passes through optional broadband ?lter 203.
`Filter 203 is used to remove large bands of undesirable
`radiation. For example. ?lter 203 can be used to remove IR
`radiation. The light then passes through a tuning assembly
`204 which passes the wavelength band of interest. Depend
`ing upon the application. assembly 204 can be as simple as
`an optical ?lter. or as complex as a continuous wavelength
`
`20
`
`25
`
`35
`
`45
`
`DESCRIPTION OF THE PREFERRED
`Ell/[BODIMENT
`FIG. 1 is an illustration of an embodiment of the inven
`tion. A sample 101 can be any of a variety of materials which
`have been treated with a ?uorochrome dye or probe. Sample
`101 can also be a sample which exhibits auto?uoresoence or
`luminescence. Sample 101 can also be a sample on which
`only a subset of the possible tests are to be performed. for
`example absorption. Sample 101 is housed in a tempaature
`controlled incubator 102.
`Sample 101 is held in place for testing by a holding ?xture
`103. A wide variety of samples can be held in ?xture 103
`with few. if any. ?xturing adjustments. For example. 6. 12.
`24. 48. 96. and 384 well microplates can be interchangeably
`used with this ?xture. Gel plates and storage phosphor plates
`can also be used with this ?xture.
`A light source 105 illuminates sample 101. If desired for
`a speci?c test. for example luminescence measurements.
`light source 105 can be deactivated Although preferably the
`wavelength range of light source 105 is from approximately
`250 nanometers (i.e.. ultraviolet radiation) to 2 micrometers
`(i.e.. infrared radiation). a smaller subset of this range is
`adequate for most present applications. Light source 105 can
`be a single source. for example a xenon arc lamp with a
`relatively ?at output from approximately 320 to 700 nanom
`eters. By changing the ?ll gas (e.g.. argon instead of xenon).
`the temperature of the ?ll gas. and the material comprising
`the lamp envelope. di?’erent wavelength bands are obtain
`able. Light source 105 can also be a laser operating at one
`or more wavelengths. To obtain a broader wavelength band.
`the output of two or more sources can be combined. Beam
`splitters or optical ?bers can be used to combine the outputs
`of the individual sources. It is possible to combine the
`outputs of the individual sources such that all sources emit
`simultaneously and. in the case of multiple laser sources.
`oo-linearly. However. in the preferred embodiment either the
`user or the system in automatic mode determines the appro
`priate wavelength or wavelength band for the selected
`application and activates the appropriate source.
`The radiation emitted by source 105 passes through a
`tuning section 107 and focussing optics 109 prior to irradi
`ating sample 101. Fluorescence and/or luminescence is
`imaged onto a detector 111 after passing through imaging
`optics 113 and a tuning section 115. Detector 111 and
`associated optics 113 and tuning section 115 can be mounted
`in a variety of locations in order to optimize performance.
`including both above and below sample 101.
`A second detector 117 is mounted below sample 101 and
`is used for absorption measurements. Detector 117 can be
`
`Agilent Exhibit 1219
`Page 10 of 17
`
`

`

`5.784.152
`
`5
`tuning system. In this embodiment. after the light passes
`through assembly 204. the light impinges on a beamsplitter
`205 which re?ects the desired wavelengths. For example.
`beamsplitter 205 may only re?ect those wavelengths nec
`essary to excite a selected ?uorochrome. The re?ected
`radiation then passes along light path 207. through condens
`ing optics 209. and impinges on sample 211. The incident
`light causes the ?uorochromes on the various probes to
`?uoresce. the emitted ?uorescence following path 213. Also
`following path 213 is light which was scattered by sample
`211. In order to accurately measure the emitted ?uorescence.
`the scattered radiation is removed. The light leaving sample
`211 and following path 213 is incident on beamsplitter 205.
`Since the re?ection coating on beamsplitter 205 is designed
`to re?ect those Wavelengths necessary for exciting the
`selected ?uorochromes while passing all other radiation.
`beamsplitter 205 removes the scattered light by re?ecting it
`away from path 213 while passing the emitted ?uorescence.
`The emitted ?uorescence is further ?ltered using ?lter 215.
`At this point the light is ready for spectral dissection and
`detection.
`In the preferred embodiment of the invention. both the
`wavelength and the bandwidth of the excitation radiation as
`well as the wavelength and the bandwidth monitored by the
`detectors are tunable. Although speci?c applications may
`require only the ability to control the wavelength of either
`the excitation or the detection subsystems. by providing
`control of both it is easy to obtain a detailed spectral analysis
`of a sample. In an alternate embodiment of the invention.
`complete tunability is only provided in one subsystem (i.e..
`excitation or detection subsystem). while course tuning (e.g..
`using a set of ?lters) is provided in the other subsystem.
`A number of techniques can be used for spectral discrimi
`nation with either the excitation or detection subsystems.
`These techniques fall into four categories: dispersive
`elements. di?=ractive elements. interferometric elements. and
`?lters.
`A prism is a dispersive element which. in its standard
`form. is non-linear as a function of deviation. This non
`linearity results in a rather complex optical apparatus design.
`‘Therefore to minimize the complexity of the optical design.
`it is preferable to use a constant deviation dispersing prism
`such as the Pellin-Broca prism shown in FIG. 3. In this type
`of prism a single monochromatic ray 301 will pass through
`the prism and exit at a deviation of 90 degrees from the
`initial incident beam 303. All other wavelengths will emerge
`from the prism at different angles. By rotating the prism
`along an axis normal to the plane of the image in FIG. 3. the
`incoming ray will have a di?'erent angle of incidence and a
`different Wavelength component will exit the prism at a
`deviation of 90 degrees. This type of prism obviously
`simpli?es the design of the apparatus since the system can
`operate at a ?xed angle and the wavelength can be tuned by
`rotating the prism.
`A grating can also be used to spectrally disperse the
`emitted ?uorescent spectra. FIG. 4 shows one con?guration
`of a wavelength di?ractive system comprising grating 401.
`folding mirror 403. entrance and exit slits 405. and aperture
`407. The wavelength is tuned by rotating grating 401. The
`bandwidth of this system is a function of the grating groove
`spacing. the aperture diameter. and the distance between the
`aperture and the grating. In the preferred con?guration of
`this embodiment multiple gratings are used which can be
`remotely selected depending upon the wavelength region of
`interest. Using multiple gratings insures that su?icient radia
`tion is collected Within all of the spectral bands of interest.
`Another approach to tuning the wavelength in either the
`excitation or detection sections of the invention is through
`
`20
`
`25
`
`35
`
`45
`
`50
`
`55
`
`6
`the use of optical ?lters. In FIG. 5 a ?lter wheel 501 contains
`a series of ?lters with a short pass edge while a ?lter wheel
`503 contains a series of ?lters with a long pass edge.
`Therefore both the Wavelength as well as the bandwidth is
`determined by the choice of ?lters. For example. by select
`ing a short pass ?lter of 450 nanometers and a long pass ?lter
`of 470 nanometers a 20 nanometer band centered at 460
`nanometers is selected. In order to insure that the wave
`length is continuously tunable. ?lter wheels 501 and 503 not
`only rotate to allow the selection of a particular ?lter. but
`they also can be rotated about axes 505. This results in the
`?lters being tilted with respect to optical axis 507. As the
`?lters are tilted off-axis their wavelength characteristics
`gradually change.
`Another approach to tuning the wavelength is to use
`variable ?lters. Circular variable ?lters are simply interfer
`ence ?lters in which the ?lm thickness varies linearly with
`the angular position on the substrate. An embodiment using
`circular variable ?lters would be similar in appearance to the
`con?guration shown in FIG. 5 except that ?lter wheels 501
`and 503 are replaced with the circular variable ?lters.
`Depending upon the position of each ?lter wheel and the tilt
`along axes 505. any Wavelength can be chosen. By control
`ling the amount of light illuminating the ?lters. through the
`use of slits. the bandwidth can also be controlled.
`In another embodiment. a Fabrey-Perot etalon tunable
`filter can be used to tune the wavelength of the excitation
`and/or detection sections of the invention. In this embodi
`ment it is generally preferable to eliminate most of the
`undesired wavelengths using a bandpass ?lter. Then the ?ne
`tuning is performed using the Fabrey-Perot system. In a
`variation of this system. ferroelectric liquid crystal devices
`can be inserted into the interference ?lters of the Fabrey
`Perot etalon. This design is capable of high throughput as
`well as rapid ?ne tuning of the system
`The preferred embodiment of the emission detection
`system is shown in FIG. 6. In this embodiment the radiation
`601 emitted by the sample is ?rst ?ltered to remove much of
`the undesired wavelength spectra using an optical ?lter 602.
`After ?ltering. radiation 601 enters a SAGNAC interfer
`ometer 603. SAGNAC interferometer 603 is comprised of a
`beam splitter 605 and turning mirrors 607. Wavelength
`selection is accomplished by controlling the optical path
`di?ierence of the interferometer. Adjustable slit 609 controls
`the bandwidth. Optics 611 focus the radiation passing
`through the interferometer and produce a real image onto
`detector 613. In this embodiment detector 613 is a CCD
`array and there is a one to one correspondence between the
`sample and the projected image of the sample.
`As illustrated in FIG. 6. beamsplitter 605 divides the
`incoming light into two separate beams. These beams are
`recombined to form an interference pattern at detector array
`613. The pattern’s intensity at each pixel of array 613 varies
`with the optical path di?‘erence. By measuring the intensity
`versus the optical path difference. an interferogram is cre
`ated. In order to recover the wavelength spectra at each pixel
`of array 613. a Fourier transform of each interferogram is
`calculated. preferably using processor 125.
`FIG. 7 illustrates a monolithic form of an interferometer
`700. The monolithic interferometer is more immune to
`vibration. misalignment. and thermal etfects then other
`interferometer con?gurations. This form of interferometer
`has a very large acceptance angle.
`Interferometer 700 is comprised of a ?rst piece of glass
`701 bonded to a second piece of glass 703 along the plane
`of a beamsplitter coating 705. Light is incident on the
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`interferometer along path 707. When this light ray hits
`bearnsplitter coating 705. the ray is split into two rays. one
`ray following path 709 and the other ray following path 711.
`After being re?ected by interferometer mirrors 713. the rays
`exit the optic along paths 715 separated by a distance 717.
`In at least one embodiment of the invention. light source
`105 can be temporarily disabled so that luminescence mea
`surements can be performed. Source 105 can either be
`disabled manually through user selection. or automatically
`by processor 125 when a luminescence test is selected. After
`disablement of source 105. reagents ?'om one or more
`reagent lines can be dispensed into sample 101 from reagent
`dispensing mechanism 127. Preferably the reagents are
`dispensed within distinct wells of a multi-well microplate.
`The time between dispensing the reagents and taking a
`reading is adjustable.
`In at least one embodiment of the invention. absorption
`measurements are made using detector 117. In this embodi
`ment a speci?c wavelength band for the excitation radiation
`is selected using tuning section 107. By measuring the
`amount of light transmitted through sample 101. the absorp
`tion characteristics of sample 101 can be determined. In
`order to achieve a wide range of measurement sensitivity a
`series of ND ?lters 121 are interposed between sample 101
`and detector 117. Preferably ND ?lters 121 are contained in
`a ?lter wheel. In one con?guration. processor 125 deter
`mines the appropriate ND ?lter based on the output of
`detector 117. In an alternate con?guration. a secondary
`detector (not shown) is placed in close proximity to detector
`117. The secondary detector is less sensitive to overexposure
`and therefore can be used to select an appropriate ND ?lter
`121. thus minimizing the risk of damaging detector 117.
`Samples 101 contained in a variety of sample containers
`can be analyzed with the present invention. FIG. 8 is an
`illustration of the cross-section of a typical microplate 801
`containing 6 sample wells 803. In a microplate of this type.
`each well 803 contains an individual specimen. After sample
`preparation. microplate 801 is placed within holding ?xture
`103. The preferred embodiment of the present invention is
`capable of utilizing microplates with 6. 12. 24. 48. 96. or 384
`wells. The preferred embodiment can also analyze gels and
`storage phosphor plates. Preferably. the user enters the
`desired sample con?guration into processor 125. Processor
`125 then deten'nines the appropriate sample reading strategy
`based on the user selected con?guration.
`The present invention is capable of analyzing sample 101
`at multiple locations within each individual sample well. In
`other words. if a 6 well microplate is selected. such as the
`microplate illustrated in FIG. 8. the user is able to obtain
`?uorescence. luminescence. and absorption information
`(depending upon the con?guration of the invention) at
`multiple locations within each sample well 803.
`In one embodiment of the invention. the user speci?es the
`sample con?guration (e.g.. a microplate with 6 wells) and
`the number of locations within each well to be tested. In this
`embodiment. data processor 125 determines the locations of
`the testing based on a predetermined test pattern. For
`example. if the user selects four sample locations and a 6
`well microplate. processor 125 would then test each sample
`well at four locations 805.
`FIG. 9 is a functional block diagram of an alternate
`embodiment in which the user can specify the actual loca
`tions within a sample well at which testing is to be per
`formed. Preferably. processor 125 is coupled to a user
`interface 901 such as a keyboard. Processor 901 is also
`coupled to a monitor 903. After the user selects a sample
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`con?guration using interface 901. a schematic representa
`tion of the selected sample con?guration is presented on
`monitor 903. The user then indicates a speci?c sample well
`to be analyzed using interface 901. Alternatively. the user
`can indicate the sample well of interest using a pointing
`device 905 (e.g.. a mouse). In the preferred embodiment.
`once a sample well has been selected. monitor 903 presents
`a magni?ed view of a single well. The magni?ed view
`makes it easier for the user to indicate the areas for mea
`surement. The user indicates the speci?c areas within the
`selected sample well which are to be analyzed using either
`interface 901 or pointing device 905. After the locations
`have been entered. the system can then be programmed to
`either analyze only the selected sample well or to use the
`same locations for measuring every sample well within the
`microplate. These locations may also be stored for later use
`with subsequent microplates.
`In