United States Patent
`Heffel?nger et al.
`
`[19]
`
`US006043506A
`Patent Number:
`Date of Patent:
`
`[11]
`[45]
`
`6,043,506
`Mar. 28, 2000
`
`[54] MULTI PARAMETER SCANNER
`
`[75] Inventors: David M. He?'el?nger, San Pablo;
`Rebecca Ann Batterson, San Rafael;
`Renato Salgado, Rodeo, all of Calif.
`
`[73] Assignee: Bio-Rad Laboratories, Inc., Hercules,
`Calif.
`
`[21] Appl. No.: 09/001,254
`
`[22]
`
`Filed:
`
`Dec. 30, 1997
`
`Related U.S. Application Data
`Provisional application No. 60/055,567, Aug. 13, 1997.
`
`[60]
`
`[51] Int. Cl.7 ..
`
`G03B 42/02; G01N 21/64
`
`[52] U.S. Cl. ....................................... .. 250/584; 250/458.1
`
`[58] Field of Search ................................... .. 250/584, 585,
`250/586, 458.1, 484.4; 356/417
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,786,813 11/1988 Svanberg et a1. .
`5,062,942 11/1991 Kambara et a1. .
`5,069,769 12/1991 Fujimiya et a1. .
`5,138,170
`8/1992 Noguchi et a1. .
`
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`
`2 024 412
`WO 90/10219
`WO 96/18205
`
`1/1980 United Kingdom .
`9/1990 WIPO .
`6/1996 WIPO .
`
`OTHER PUBLICATIONS
`
`T. Reid, et al., “Simultaneous Visualization of Seven Dif
`ferent DNA Probes by in situ Hybridization Using Combi
`natorial Fluorescence and Digital Imaging Microscopy”
`Proc. Natl. Acad. Sci, USA, (Feb. 1992) vol. 89, pp.
`1388—1392.
`
`R.M. Cothren, et al., “Gastrointestinal Tissue Diagnostics by
`Laser—Induced Fluorescence Spectroscopy at Endoscopy,”
`Gastrointestinal Endoscopy, vol. 36, No. 2, (Mar/Apr.
`1990), pp. 105—111.
`(List continued on next page.)
`
`Primary Examiner—EdWard P. Westin
`Assistant Examiner—Richard Hanig
`Attorney, Agent, or Firm—David G. Beck; ToWnsend and
`ToWnsend and CreW, LLP
`[57]
`ABSTRACT
`
`An apparatus capable of measuring quantities of biological
`or other types of samples that have been labeled using any
`of a variety of techniques including ?uorescence,
`radioisotopes, enzyme activated light emitting chemicals,
`and enzyme activated ?uorescent materials is provided. The
`apparatus alloWs for either simultaneous or sequential acqui
`sition of signals from multiple sample types. The apparatus
`is not restricted to a particular source or Wavelength of
`excitation or readout light, nor is the apparatus restricted to
`a particular emission Wavelength. The provided scanner
`includes a source module that preferably contains an internal
`laser emitting tWo different Wavelengths of approximately
`the same intensity. An optional external light source may be
`coupled to the source module, thus adding further ?exibility
`through the addition of other Wavelengths (e.g., V, visible,
`mid-IR, and IR). The scanner also includes a detection
`module. Within the detection module are tWo detectors, thus
`alloWing the simultaneous detection of multiple Wave
`lengths. A bifurcated optical cable is used to transfer the
`excitation and/or readout light from the source module to the
`sample and subsequently transfer the emitted and/or scat
`tered light from the sample to the detection module. The
`scanning stage of the scanner is designed to accommodate a
`variety of samples, ranging from phosphor screens, gels, and
`?uorescent samples to microtiter plates. An internal micro
`processor is used to control the various aspects of the
`scanner, preferably including translation stage control,
`source ?lters, and detection ?lters. The internal micropro
`cessor may be coupled to an external computer. The external
`computer may be used to change the programming of the
`microprocessor, provide a user interface to the
`microprocessor, process and store test results, and display
`sample images.
`
`45 Claims, 7 Drawing Sheets
`
`SAMPLE
`TRAY
`
`EXTERNAL
`COMPUTER
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`6,043,506
`Page 2
`
`US. PATENT DOCUMENTS
`
`OTHER PUBLICATIONS
`
`5,190,632
`5,213,673
`5,246,866
`5,266,803
`5,290,419
`5,424,841
`5,436,718
`5,459,325
`5,461,240
`5,528,050
`5,578,818
`5,591,981
`5,780,857
`
`3/1993
`5/1993
`9/1993
`11/1993
`3/1994
`6/1995
`7/1995
`10/1995
`10/1995
`6/1996
`11/1996
`1/1997
`7/1998
`
`Fujimiya et a1. .
`Fujimiya et a1. .
`Nasu et a1. .
`
`Heffel?nger .
`Kambara et a1. .
`Van Gelder et a1. .
`Fernandes et a1. .
`
`Hueton et a1. .
`
`Karasawa .............................. .. 250/585
`Miller et a1. .
`Kain et a1. .
`Heffel?nger et a1. .
`Harju et a1. .
`
`P.S. Anderson, et al., “Auto?uorescence of Various Rodent
`Tissues and Human Skin Tumour Samples,” Lasers in
`Medical Science, vol. 2, No. 1 (Jan.—Mar. 1987), pp. 41—49.
`J..Z. Sanders, et al., “Imaging as a Tool for Improving
`Length and Accuracy of Sequence Analysis in Automated
`Fluorescence—Based DNA Sequencing,” Electrophoresis
`No. 12, (1991), pp. 3—11.
`Product Literature for STORM Gel and Blot Imaging Sys
`tem produced by Molecular Dynamics. @1995 Molecular
`Dynamics, Inc.
`Product Catalogue for Life Science Solutions produced by
`Molecular Dynamics. @1995 Molecular Dynamics, Inc.
`
`THERMO FISHER EX. 1015
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`Sheet 3 0f 7
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`1
`MULTI PARAMETER SCANNER
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a Continuation-In-Part of US. Provi
`sional application Ser. No. 60/055,567 ?led Aug. 13, 1997,
`the complete disclosure of Which is incorporated herein by
`reference for all purposes. This application is related to
`commonly assigned US. Pat. Nos. 5,591,981, issued Jan. 7,
`1997 and 5,266,803, issued Nov. 30, 1993 and to commonly
`assigned, US. patent application Ser. Nos. 08/585,303, ?led
`Jan. 11, 1996 now US. Pat. No. 5,863,504, 08/729,111, ?led
`Oct. 11, 1996 now US. Pat. No. 5,784,152, and to 08/927,
`556, ?led Sep. 9, 1997 now US. Pat. No. 5,885,531, the
`complete disclosures of Which are incorporated herein by
`reference for all purposes.
`
`10
`
`15
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to optical scanners
`and, more particularly, to a method and apparatus for mea
`suring and/or imaging biological or other types of samples
`that have been labeled using a variety of techniques.
`
`20
`
`BACKGROUND OF THE INVENTION
`
`Imaging is an important tool used in the detection of a
`variety of biological molecules. For example, imaging
`devices may be used to detect and determine concentrations
`of molecules of a speci?c molecular Weight, DNA, a speci?c
`DNA sequence, proteins, and carbohydrates. Typically the
`samples of interest are labeled using ?uorescent dyes,
`radioisotopes, or enZyme activated light emitting (i.e.,
`chemiluminescent) or ?uorescent (i.e., chemi?uorescent)
`chemicals.
`UV, visible or IR light excites ?uorescent dyes and
`markers. Once excited the dyes ?uoresce, preferably emit
`ting light at a Wavelength distinguishable from the excitation
`Wavelength. Radioactive and chemiluminescent signals are
`typically captured using either x-ray ?lm or storage phos
`phor screens. The x-ray ?lm is developed and read using a
`densitometer. The storage phosphor screen does not require
`development and is read out by scanning the screen With a
`beam of light. The readout beam produces an emission from
`the storage phosphor, the intensity of the emission being
`proportional to the original quantity of radiation retained by
`the storage phosphors.
`A variety of devices have been described for use in
`detecting labeled biological samples. US. Pat. No. 3,746,
`840 discloses a device for high-resolution readout of infor
`mation stored on a ?lm. The device comprises a slit equal in
`Width to the desired resolution With optical ?bers behind the
`slit of a diameter equal to the slit Width. The optical ?bers
`collect the light as it crosses the slit and transmits it to the
`detectors.
`US. Pat. No. 3,836,225 discloses a ?ber optic laser
`scanner. The disclosed scanner uses tWo optical ?ber sets
`attached to electromagnetic coils. The magnetic coils de?ect
`the beam as required.
`US. Pat. No. 3,892,468 discloses a passive array of
`variable length optical ?bers that function as a dynamic
`scanner. Each consecutive ?ber in the ?ber array is incre
`mentally longer than the preceding ?ber. Thus light entering
`the ?bers at the same time Will exit the ?bers at different
`times, the variations in exit times thus being correlated With
`different locations.
`US. Pat. No. 4,877,966, discloses a device for measure
`ment of loW-level laser induced phosphorescence. The laser
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`is directed through a beam expander and then aimed by
`mirrors. The induced phosphorescence is collected by a ?ber
`optic face plate and passed to a photomultiplier tube.
`US. Pat. No. 5,062,942 discloses a ?uorescence detection
`system for use With electrophoresis gel plates. In the dis
`closed system the gel plate is illuminated With a laser
`excitation source and the emitted ?uorescent light is sepa
`rated into a plurality of virtual images that are subsequently
`passed through individual bandpass ?lters thereby providing
`multicolor ?uorescence detection.
`US. Pat. No. 5,290,419 discloses a multicolor ?uores
`cence detection system utiliZing multiple laser sources and
`means for detecting ?uorescence as a function of Wave
`length. The individual laser sources are combined With a
`light chopper (e.g., rotary shutter) in order to irradiate the
`sample on a time-sharing basis.
`US. Pat. No. 5,436,718 discloses a multi-function pho
`tometer for measuring the absorbance, ?uorescence, and
`luminescence associated With a sample. The disclosed sys
`tem uses optical ?bers to transmit light to and from the
`sample using scanning head. A computer controlled posi
`tioning table is used to position the canning head With
`respect to the samples contained in a microplate.
`US. Pat. No. 5,459,325 discloses a high-speed ?uores
`cence scanner. The system utiliZes a lightWeight scan head
`to scan a collimated excitation beam across the sample. The
`emitted ?uorescence is gathered by the scan head lens and
`directed back along the optical path of the excitation beam
`to a detector. In order to obtain a tWo-dimensional image of
`the sample, the sample is translated in an axis orthogonal to
`the scan line.
`In a publication entitled Imaging as a Tool for Improving
`Length and Accuracy of Sequence Analysis in Automated
`Fluorescence-Based DNA Sequencing by Sanders et al, a
`method of signal analysis is disclosed. (Electrophoresis
`1991, 12, 3—11). In the disclosed method, a computer
`program Was used to remove distortions in the DNA bands
`in sequencing gels, thus improving the accuracy of DNA
`sequence analysis. The authors noted that the disclosed
`techniques should be applicable to other systems such as gel
`electrophoresis of proteins and DNA restriction fragments.
`The scanners described above do not take full advantage
`of the Wide range of different sample types available. Rather,
`a typical scanning device is designed for a speci?c type of
`sample, e.g., ?uorescent samples, and as a result is incapable
`of use With another type of sample. In addition, many
`biological sample scanners offer a very limited set of
`irradiation/excitation Wavelengths and/or emission
`Wavelengths, thus further limiting the functionality of the
`device. Lastly, the resolution offered by many, if not all, of
`the fore-mentioned markers is not fully utiliZed by most
`biological sample scanning systems.
`Therefore a compact optical scanner capable of use With
`a variety of sample types and con?gurations that offers
`multiple excitation/irradiation Wavelengths and that may be
`used to detect emissions at a variety of Wavelengths is
`desirable.
`
`SUMMARY OF THE INVENTION
`The present invention provides an apparatus capable of
`measuring quantities of biological or other types of samples
`that have been labeled using any of a variety of techniques
`including ?uorescence, radioisotopes, enZyme activated
`light emitting chemicals, and enZyme activated ?uorescent
`materials. The apparatus alloWs for either simultaneous or
`sequential acquisition of signals from multiple sample types.
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`The apparatus is not restricted to a particular source or
`Wavelength of excitation or readout light, nor is the appa
`ratus restricted to a particular emission Wavelength. Thus the
`present invention is capable of measuring every type of
`?uorescent dye, storage phosphor screen, and chemilumins
`cent probe.
`In one aspect of the invention, the scanner includes a
`source module. The source module has an internal laser that
`emits tWo Wavelengths, 532 nanometers and 1064
`nanometers, of approximately the same intensity. These tWo
`Wavelengths alloW the scanner to function With storage
`phosphor screens based on BaFBrzEu, SrS:Ce, and SrS:Sm
`as Well as a variety of ?uorescent dyes and other stains. An
`optional external light source may be easily coupled to the
`source module, thus adding further ?exibility to the scan
`ner’s potential applications through the addition of other
`Wavelengths in the UV, visible, mid-IR, and IR spectral
`ranges. The external light source passes through a beam
`splitter that combines the emissions from the internal laser
`With those of the external source(s). The light emitted by the
`external source undergoes an auto-alignment procedure to
`insure optimal coupling betWeen the source and the optical
`system of the scanner.
`In another aspect of the invention, the scanner includes a
`detection module. Within the detection module are tWo
`detectors, thus alloWing the simultaneous detection of mul
`tiple Wavelengths. A variety of bandpass ?lters and beam
`splitters contained in at least tWo ?lter Wheels provide the
`means of removing undesired radiation from the light beam
`prior to detection. Preferably the tWo detectors are photo
`multiplier tubes, thus providing high sensitivity over a
`relatively Wide Wavelength range.
`A bifurcated optical cable is preferably used to transfer
`the excitation and/or readout light from the source module to
`the sample and subsequently transfer the emitted and/or
`scattered light from the sample to the detection module.
`Although neither the number nor the physical arrangement
`of the ?bers is critical, typically betWeen 1 and 10 excitation
`?bers are surrounded by betWeen 100 and 300 collection
`?bers in order to form the scanning head probe. Coupled to
`the end of the ?ber scanning probe are focussing optics and
`condensing optics. In order to accommodate a range of
`sample siZes Without adjusting the separation distance
`betWeen the probe and the sample, preferably the probe
`optics provide a focal spot siZe of less than 150 micrometers
`over a 5 millimeter range. Alternatively, either the scanning
`head probe or the optics Within the probe may be coupled to
`a translation stage, thus alloWing the scanning probe to be
`optimiZed for different sample siZes.
`In another aspect of the invention, the system includes a
`scanning stage for scanning the probe across the sample. The
`system is designed to accommodate a variety of samples and
`sample types, ranging from phosphor screens, gels, and
`?uorescent samples to microtiter plates. The scan head is
`mounted to a pair of translation stages, thus alloWing the
`probe to scan the entire available sampling area or some
`subset thereof In one embodiment of the invention, the
`scanning system operates in a closed loop fashion, thereby
`providing direct position feedback. Positional information
`may be obtained using optical encoders, either mounted
`Within the motors operating the translation stages or
`mounted in such a Way as to monitor stage travel of the
`individual translation stages.
`In another aspect of the invention, a microprocessor
`controls the scanning system. In one embodiment the micro
`processor controls motors coupled to the scanner’s transla
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`tion stages, thus alloWing the microprocessor to control the
`scan speed as Well as the sampling area of the scanner. In
`another embodiment the microprocessor also controls the
`?lter Wheels in the source module and the ?lter Wheels in the
`detector module. In yet another embodiment the micropro
`cessor controls the high voltage supplies for photomultiplier
`tube detectors in the detection module, thus alloWing the
`gain of the detectors to be varied depending upon the
`requirements imposed by the sample.
`The microprocessor of the present invention may be
`coupled to an external computer. The external computer may
`be used to change the programming of the microprocessor,
`thus alloWing the system to be altered as different detector
`modules, source modules, and external sources are added to
`the system. The external computer may also be used to
`provide the user With a means of programming the micro
`processor for a speci?c test run, for example, for a speci?c
`sample type and siZe. In order to simplify programming,
`either the microprocessor or the external computer may
`include a look-up table containing a variety of operating
`parameters and/or programming instructions based on the
`intended conditions of operation (e.g., sample type, irradia
`tion Wavelengths, detection Wavelengths, etc.). The external
`computer may also be used for test result storage as Well as
`providing a means of processing and displaying the test
`results. The results may be displayed in a variety of formats,
`including tabular and sample image displays. The external
`computer may also be used to present the data in a manner
`that is more understandable by the user, for example, rep
`resenting different emittance intensities or Wavelengths by
`different colors.
`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 schematically illustrates an overvieW of a scanner
`according to the present invention;
`FIG. 2 is an illustration of the preferred embodiment of
`the source;
`FIG. 3 is an illustration of the preferred embodiment of
`the detection system;
`FIG. 4 is an illustration of the scanning mechanism of the
`preferred embodiment;
`FIG. 5 is an expanded vieW of the preferred embodiment
`of the scan head;
`FIG. 6 illustrates the cross-section of the preferred
`embodiment of the optical ?ber bundle;
`FIG. 7 illustrates the upper and loWer enclosures for the
`preferred embodiment of the invention;
`FIG. 8 illustrates an end vieW of the loWer housing
`enclosure shoWn in FIG. 7;
`FIG. 9 illustrates one embodiment of a sample holding
`tray;
`FIG. 10 illustrates an embodiment of a sample holding
`tray that includes a light cover;
`FIG. 11 illustrates a cross-section of a portion of a storage
`phosphor exposure platform;
`FIG. 12 illustrates an outer vieW of the storage phosphor
`exposure system shoWn in FIG. 11; and
`FIG. 13 illustrates an upper cross-sectional vieW of the
`storage phosphor exposure system shoWn in FIG. 11.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`FIG. 1 schematically illustrates an overvieW of a scanner
`according to the present invention. Although a variety of
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`external components may be attached to the system for
`added versatility, the principal system components are
`designed to ?t Within a compact, lightweight assembly 101.
`The sample of interest is placed on a sample tray 103 Within
`assembly 101. Sample tray 103 is con?gured to hold a
`variety of sample types, thus adding to the versatility of the
`device. A scanner head 105 is movably coupled to a pair of
`translation members 107 and 109. Translation members 107
`and 109 alloW scanner head 105 to be scanned over the
`entire sample or any portion thereof.
`An optical means 111 is coupled to scanner head 105,
`thereby alloWing radiation from a source 113 to pass through
`scanner head 105 and impinge on a small, selected area of
`the sample held in sample holder 103 Preferably optical
`means 111 is comprised of a ?ber optic, thus providing a
`simple means of coupling energy from source 113 to scan
`ning head 105. Light emitted and/or scattered by the sample
`is collected at head 105, passed through optical means 111,
`and detected by a detection system 115. Alternatively, light
`passing through the sample and re?ected from a re?ective
`surface placed beloW the sample may be collected at head
`105, passed through optical means 111, and detected by
`detection system 115, thus yielding a quantity that may be
`correlated to the absorption of the sample. Alternatively, the
`light re?ected by the sample may be collected at head 105,
`passed through optical means 111, and detected by detection
`system 115. The means for coupling head 105 to source 113
`may be different from the means for coupling head 105 to
`detection system 115, hoWever preferably a bifurcated ?ber
`optic is used such as that disclosed in US. Pat. No. 5,266,
`803, the disclosure of Which is incorporated herein in its
`entirety.
`A microprocessor 117, coupled to translation stages 107
`and 109, is used to control the scanning operation, for
`example the scan speed. Microprocessor 117 is also coupled
`to source 113 and detection system 115. Although micro
`processor 117 may be con?gured to independently operate
`the scanning system, it may also be coupled to an external
`computer system 119. External computer 119 may be used
`to program processor 117, monitor experimental progress,
`store test results, and construct and display sample images
`from the signals detected by system 115. External computer
`119 may also be used in conjunction With processor 117 to
`control and manipulate the scanning process and the result
`ant data (e.g, automatic lane ?nding, automatic band ?nding,
`automatic quantitation of results, user-de?ned templates for
`automatic quantitation parameters, color correction, tiling
`memory management, etc.).
`Although source 113 may be any of a variety of source
`types (e.g., laser, continuously tunable broadband source,
`etc.), the preferred embodiment of source 113 is illustrated
`in FIG. 2. Within source 113 is a dedicated laser 201
`producing multiple Wavelengths. The output intensity of
`laser 201 may be controlled by neutral density ?lters or by
`digitally controlling the poWer supply for the laser. Prefer
`ably laser 201 is a diode pumped solid state laser emitting
`light at 532 nanometers and at 1064 nanometers. The dual
`Wavelength capabilities of laser 201 alloW a Wide range of
`samples to be excited Without requiring any changes to the
`system. In the preferred embodiment, either the cavity
`mirrors of laser 201 or the coatings of the laser optics are
`designed such that the laser emits approximately the same
`energy intensity Within the tWo selected Wavelengths. By
`providing approximately the same output poWer, multiple
`types of phosphor screens can be ef?ciently scanned Without
`requiring adjustments to the system. For example, storage
`phosphor screens based on BaFBrzEu, SrSzCe, and SrSzSm
`may all be used With this source.
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`The light emitted by laser 201 is re?ected by a beam
`combining mirror 203 into focussing assembly 205. Mirror
`203 is designed to ef?ciently re?ect both of the Wavelengths
`emitted by laser 201. Assembly 205 focuses the re?ected
`beam onto the end of ?ber 207. Micro-positioners 209 and
`211, coupled to laser 201 and mirror 203 respectively, are
`used to accurately locate the laser beam onto ?ber 207, thus
`achieving the optimal transfer of energy from laser 201
`through ?ber 207 to the sample. Once laser 201 and mirror
`203 have been properly located and locked into position,
`repositioning of these components is only required if one of
`them is inadvertently moved or if the laser beam exiting
`laser 201 exhibits movement as the laser ages. HoWever the
`preferred embodiment of the system is designed to minimiZe
`if not altogether eliminate the need for positional adjustment
`by the user.
`In order to provide additional ?exibility as Well as the
`potential for use With as-yet undiscovered samples and
`targets, the preferred embodiment of source 113 provides for
`an external source 212. External source 212 is coupled to the
`scanning system through an external port 213. The light
`from the external source passes through port 213 to a
`collimating assembly 215. Collimating assembly 215 colli
`mates the light from external source 212 and passes the
`collimated light through beam combining mirror 203 and
`focussing assembly 205 into ?ber 207. The optical coatings
`on mirror 203 are designed to maximiZe re?ection at the
`desired Wavelengths emitted by laser 201 While simulta
`neously maximiZing transmittance of all other Wavelengths,
`particularly the Wavelengths of potential interest for an
`external source. Thus multiple excitation Wavelengths may
`be simultaneously transmitted through ?ber 207 to the
`sample, i.e., dual Wavelengths from laser source 201 and one
`or more Wavelengths from one or more external source(s)
`212.
`A variety of external sources 212 may be coupled to
`external port 213. Both lasers and broadband sources may be
`coupled into the scanning system, depending upon the
`desired Wavelength(s). Generally, the external source may
`be any source of ultraviolet (i.e., UV), visible, near infrared
`(i.e., NIR), or infrared (i.e., IR) radiation. Thus the external
`source may be continuously tunable or not, pulsed or
`continuous, coherent or incoherent, and be in the form of a
`laser or an arc lamp or some other source emitting the
`desired radiation.
`As discussed above, dual Wavelength internal laser source
`201 may be used With storage phosphor screens based on
`BaFBrzEu, SrSzCe, and SrSzSm. In addition, internal source
`201 may be used With a variety of dyes, stains, ?uorescent,
`and chemiluminescent markers, depending upon the
`required excitation Wavelength. Potential dyes for use With
`the 532 nanometer line, and therefore not requiring an
`external source, include the folloWing ?uorescent dyes; JOE,
`TAMRA, ROX, HEX, Bodipy, TRITC, CY3, Rhodamine B,
`and Lissamine Rhodamine. In addition, this Wavelength
`laser line may be used to excite DNA stains based on
`Ethidium Bromide, Ethidium homodimer, POPO-3, Radiant
`Red as Well as protein stain Sypro Red. Additionally a
`variety of external sources may be coupled to port 213 and
`used With a variety of dyes/stains. For example, the 488
`nanometer line emitted by Argon and Argon/Krypton lasers
`may be coupled to external port 213 and potentially used
`With ?uorescent dyes (e.g., FAM, Bodipy FL, Lucifer
`YelloW, NBD-X Nile Red, Oregon Green, CY2, TET, HEX
`R6G, JOE, and FITC), SS stains (e.g., SYBR Green II,
`Radiant Red, YOYO-l, and TOTO-1), protein dyes (e.g.,
`Nile Red and SYPRO Orange), and DNA stains (e.g., Pico
`
`THERMO FISHER EX. 1015
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`6,043,506
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`7
`Green, Vistra Green, SYBR Green I, YOYO-l, and TOTO
`1). TWo other Well known dyes, CY5 and CY7, require
`excitation in the 650 nanometer range and therefore a
`potentially suitable laser is an Argon/Krypton laser emitting
`at the 647 line. Other potential laser sources include HeNe
`lasers, operating either in the red or green, and frequency
`doubled YAG lasers. This list of potential external sources
`matched to various dyes and stains is intended for illustra
`tive purposes only, and is not intended to be exhaustive. The
`design of the present invention is such that the number and
`type of different sources that may be coupled through port
`213 into the scanner is practically limitless.
`External port 213 typically does not provide suf?cient
`precision to optimally couple external source 212 to the
`scanning system. Therefore preferably a translation stage
`system 217 is coupled to collimating assembly 215 thus
`alloWing the emission from external source 212 to be
`optimally coupled to the scanner. Although stage 217 may be
`manually operated, preferably stage 217 is controlled by
`microprocessor 117, thereby alloWing for auto-alignment of
`the optical system.
`A variety of alignment algorithms may be used to opti
`miZe the optical throughput of the external source. The
`alignment may be performed on a periodic basis, prior to
`each scan, or only after the initial coupling of external
`source 212 to port 213. Basically translation stage 217 must
`be moved until the maximum amount of energy from
`external source 212 passes through collimating assembly
`215 and into focussing assembly 205. In one embodiment of
`the invention, stage 217 initially undergoes a rough adjust
`ment feedback loop simply to ?nd the general preferred
`location of collimator 215. FolloWing the rough adjustment,
`a ?ne adjustment feedback loop determines the optimum
`stage location. The auto-alignment procedure may be as
`simple as moving the stage in prede?ned incremental steps
`in a raster scanning fashion While recording the coupling
`ef?ciency at each step. After the raster scan is complete, the
`stage may be moved back to the location offering the highest
`ef?ciency and the raster scan can then be repeated using
`smaller incremental steps. Although this process may be
`repeated numerous times, in the preferred embodiment a
`single rough scan folloWed by a single ?ne scan has been
`determined to be adequate. In order to minimiZe the storage
`capacity used to store the coupling ef?ciency noted for each
`position of stage 217, the system may be programmed to
`discard coupling efficiencies beloW a prede?ned ef?ciency.
`Alternatively, the system may be designed to discard cou
`pling ef?ciency samples that fall suf?ciently beloW a previ
`ously monitored coupling ef?ciency.
`Several different methods of monitoring the coupling
`ef?ciency of external source 212 to ?ber 207 and ultimately,
`the sample, may be used. In one embodiment scanning head
`105 is moved to a portion of the scanning module that
`contains a dedicated detector 219. As stage 217 is adjusted,
`the energy falling on detector 219 is monitored thereby
`providing feedback on the coupling of the external source to
`the optical system. In another embodiment, a calibration
`detector 221 may be located directly on scan head 105. A?ip
`mirror or a stationary mirror may be used to couple some
`portion of the output of ?ber 207 to detector 221. In the
`preferred embodiment, detection system 115 is used. In this
`embodiment preferably scan head 105 is ?rst moved to a
`calibration site 223 on the scanning table, thus insuring that
`sufficient energy passes through the entire assembly to
`detector 115 to alloW optimiZation of stage 217. Calibration
`site 223 may be a simple broad band re?ector, thus re?ecting
`the energy from external source 212 back through ?ber 111
`to detector 115.
`
`10
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`15
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`25
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`35
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`45
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`55
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`65
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`8
`Source 113 also contains a ?lter Wheel 225. Filter Wheel
`225 contains numerous ?lters, the selection of Which is
`provided by rotating the Wheel. Preferably ?lter Wheel 225
`is coupled to microprocessor 117, thus alloWing further
`automation of the system. The ?lters Within Wheel 225
`typically are used to limit the radiation passing through ?ber
`207 to the sample and possibly being scattered to the
`detection system. For example, although laser 201 prefer
`ably emits radiation at the desired dual Wavelengths of 532
`and 1064 nanometers, it may also emit minor amounts of
`radiation at various other Wavelengths, e.g., laser harmonics.
`These harmonics may impact the performance of the
`scanner, for example by being mistaken by detection system
`115 as emissions from the sample thereby providing an
`erroneous signal. A ?lter Within ?lter Wheel 225 may be used
`to block such laser harmonics. Besides containing ?lters,
`?lter Wheel 225 may also contain neutral density ?lters to
`control the intensity of the source as Well as an opaque
`member for use as an optical shutter. The opaque member
`Would alloW the sy