(12) United States Patent
`Giebeler et al.
`
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US006316774Bl
`US 6,316,774 Bl
`Nov. 13, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) OPTICAL SYSTEM FOR A SCANNING
`FLUOROMETER
`
`(75)
`
`Inventors: Robert Giebeler, San Jose; David G.
`Ogle, Los Altos; Roger Kaye,
`Mountain View, all of CA (US)
`
`(73) Assignee: Molecular Devices Corporation,
`Sunnyvale, CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 09/274,753
`
`(22) Filed:
`
`Mar. 23, 1999
`
`Related U.S. Application Data
`(60) Provisional application No. 60/096,999, filed on Aug. 18,
`1998.
`Int. CI.7 ..................................................... GOIN 21/64
`(51)
`(52) U.S. CI . ..................................... 250/458.1; 250/459.1;
`250/461.1
`(58) Field of Search .............................. 250/458.1, 461.1,
`250/459.1, 461.2
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`* 7/1984
`2/1985
`12/1986
`9/1987
`7/1990
`7/1992
`1/1997
`7/1998
`3/2000
`
`...................... 250/458.1
`
`Lucht et al.
`Nelson.
`Landa.
`Nebe et al. ....................... 250/458.1
`Bowen et al. .................... 250/461.1
`Umetsu et al.
`........................ 422/64
`Heffelfinger et al. .
`Heffelfinger et al. .
`Harju ..................................... 422/52
`
`4,461,573
`4,501,970
`4,626,684
`4,691,110 *
`4,945,250 *
`5,133,936 *
`5,591,981
`5,784,152
`6,042,785 *
`
`WO 97/11352
`
`3/1997 (WO) .
`
`* cited by examiner
`
`Primary Examiner-Constantine Hannaher
`Assistant Examiner-Otilia Gabor
`(74) Attorney, Agent, or Firm-McCutchen, Doyle, Brown
`& Enerson, LLP; David G. Beck
`
`(57)
`
`ABSTRACT
`
`A method and apparatus for determining the fluorescence,
`luminescence, or absorption of a sample is provided. The
`sample may either be contained within a cuvette or within
`one or more sample wells within a multi-assay plate. A
`combination of a broadband source, a monochromator, and
`a series of optical filters are used to tune the excitation
`wavelength to a predetermined value within a relatively
`wide wavelength band. A similar optical configuration is
`used to tune the detection wavelength. An optical scanning
`head assembly is used that includes mirrored optics for
`coupling the excitation source to the sample and the emitted
`light to the detector. An elliptical focussing mirror is used to
`magnify and focus the light projected from an optical fiber
`coupled to the source subassembly onto the sample. A
`portion of the source light is reflected by a beamsplitter onto
`a reference detector used to monitor the output of the source.
`The light from the elliptical mirror passes through an
`aperture in a second elliptical mirror prior to impinging upon
`the sample. The light emitted by the sample within the
`sample well is reflected by the second elliptical mirror and
`imaged onto the entrance aperture of an optical fiber coupled
`to the detector subassembly. The optical axes of both mirrors
`are slightly offset from the sample well normal. The mirror
`offset minimizes the amount of light reflected from the
`meniscus of the sample or the bottom surface of the sample
`well that enters the detection subassembly.
`
`FOREIGN PATENT DOCUMENTS
`
`WO 97/11351
`
`3/1997 (WO) .
`
`32 Claims, 26 Drawing Sheets
`
`Agilent Exhibit 1240
`Page 1 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 1 of 26
`
`US 6,316,774 Bl
`
`108
`
`123
`113
`
`129
`
`111
`
`137
`
`1001
`
`135
`
`FIG.
`
`I.
`
`Agilent Exhibit 1240
`Page 2 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 2 of 26
`
`US 6,316,774 Bl
`
`219
`
`D
`
`err r~
`r ri
`
`r::..------
`
`rr
`r
`
`CJ =
`
`FIG. 2.
`
`o
`
`,~co
`
`213
`
`ODO
`
`Agilent Exhibit 1240
`Page 3 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 3 of 26
`
`US 6,316,774 Bl
`
`.,-131
`
`101.-/
`
`315
`
`C .
`
`FIG. 3.
`
`Agilent Exhibit 1240
`Page 4 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 4 of 26
`
`US 6,316,774 Bl
`
`108
`
`107
`
`107(}§'
`FIG. 4.
`
`FIG. 5.
`
`Agilent Exhibit 1240
`Page 5 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 5 of 26
`
`US 6,316,774 Bl
`
`601
`
`603
`
`FIG. 6
`
`FIG 7.
`
`FIG. 8.
`
`Agilent Exhibit 1240
`Page 6 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 6 of 26
`
`US 6,316,774 Bl
`
`,,-331
`
`140
`
`FIG. 9.
`
`139
`
`1001
`
`FIG. IQ
`
`Agilent Exhibit 1240
`Page 7 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 7 of 26
`
`US 6,316,774 Bl
`
`/1107
`1103
`
`1105
`
`FIG.
`
`II.
`
`Agilent Exhibit 1240
`Page 8 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 8 of 26
`
`US 6,316,774 Bl
`
`FIG. 12.
`
`F/6, IJ
`
`Agilent Exhibit 1240
`Page 9 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 9 of 26
`
`US 6,316,774 Bl
`
`0
`
`1a
`I
`clJ
`I
`
`I
`I
`
`I
`1411:
`
`I
`
`I
`I
`
`I
`
`1111
`
`FIG. 14.
`
`Agilent Exhibit 1240
`Page 10 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 10 of 26
`
`US 6,316,774 Bl
`
`FIG. 15.
`
`Agilent Exhibit 1240
`Page 11 of 42
`
`

`

`i,-
`~
`,I;;..
`-...,l
`~
`O'I
`i,-
`~
`O'I
`rJ'J.
`
`e
`
`O'I
`N
`
`"'"" 0 ....,
`"'""
`~
`
`'JJ. =(cid:173)~
`
`"'""
`C
`C
`N
`"'"" ~~
`~
`z 0
`
`~ = .....
`~ .....
`~
`r:JJ. .
`d .
`
`FIG. 16.
`
`ASSEMBLIES
`CARRIAGE
`SCANNING
`
`SHUTTERS
`
`MULTIPLE FIBER
`
`OF RFU
`
`CALCULATION
`
`& OPTICAL FILTERS
`MONOCHROMATOR
`
`DETECTOR
`
`& OPTICAL FILTERS
`MONOCHROMATOR
`
`SOURCE
`
`FLASH LAMP
`
`TRIGGER
`
`PROCESSOR
`
`HV SUPPLY
`
`103
`
`SIGNAL
`
`PMT
`
`DAC
`
`1601
`
`1603
`
`~---_._,
`
`ADC
`
`MULTIPLEXER
`
`GAIN
`
`1613
`
`1611
`
`HOLD
`
`SAMPLE &
`
`INTEGRATOR
`
`PREAMP/
`
`PMT
`
`1609
`
`135
`
`1607
`
`Agilent Exhibit 1240
`Page 12 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 12 of 26
`
`US 6,316,774 Bl
`
`SELECT EMISSION
`WAVELENGTH
`
`SCAN EXCITATION
`WAVELENGTH
`
`'
`'
`
`~ -
`
`V 1701
`
`v- 1703
`
`1,,.-- 1705
`
`v 1707
`
`v 1709
`
`,,.--1711
`
`v 1712
`
`1,,.-- 1713
`
`DETERMINE PEAK
`EXCITATION WAVELENGTH
`t
`FIX EXCITATION
`WAVELENGTH AT PEAK
`+
`SCAN EMISSION
`WAVELENGTH
`•
`DETERMINE PEAK EMISSION v 1710
`WAVELENGTH
`+
`'
`'
`'
`
`SELECT EMISSION FILTER
`
`FIX EMISSION FILTER
`
`FIX EMISSION WAVELENGTH
`
`READ SAMPLES BY END v 1715
`POINT OR KINETIC MODE
`
`FIG. 17.
`
`Agilent Exhibit 1240
`Page 13 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 13 of 26
`
`US 6,316,774 Bl
`
`1801
`USER SELECTS TEST
`PARAMETERS
`1803
`DETERMINE UPPER
`PMT VOLTAGE
`1805
`SELECT MEDIUM AND
`LOW PMT VOL TAG ES
`
`13/26
`
`MEASURE PMT
`SIGNAL FOR HIGH
`REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`COEFFICIENT
`
`1807
`
`1809
`
`YES
`
`1813
`
`w= n
`
`READ SAMPLE WELL
`w WITH SINGLE FLASH
`
`w= n
`1835,........i...---'-------,
`READ SAMPLE WELL w
`~ - - -~
`~ - - -~ WITH PRESELECTED
`NUMBER OF FLASHES
`
`1819
`TAG WELL
`LOCATION
`
`1823
`
`w=n+1
`
`1827
`PROCESS
`DATA
`
`1833
`PROCESS
`DATA
`
`1837
`
`TAG WELL
`LOCATION
`
`w=n+1
`
`PROCESS
`DATA
`
`FIG. 18.
`
`Agilent Exhibit 1240
`Page 14 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 14 of 26
`
`US 6,316,774 Bl
`
`USER SELECTS TEST
`PARAMETERS
`
`USER SELECTS PMT
`VOLTAGE
`
`MEASURE PMT SIGNAL
`FOR HIGH REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`COEFFICIENT
`
`1801
`
`1901
`
`1807
`
`1809
`
`w=n
`~ - - . . . , _ _ _ ___.___,
`1815
`READ SAMPLE WELL ~------.
`w WITH SINGLE FLASH
`
`1819
`
`TAG WELL
`LOCATION
`
`1823
`
`w=n+1
`
`PROCESS DAT A
`
`YES
`
`1813
`
`w= n
`
`1835
`READ SAMPLE WELL
`wWITH
`PRESELECTED
`NUMBER OF FLASHES
`1837
`AVERAGE DATA
`
`1819
`TAG WELL YES
`LOCATION
`
`w=n+1
`
`PROCESS DAT A
`
`FIG. 19.
`
`Agilent Exhibit 1240
`Page 15 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 15 of 26
`
`US 6,316,774 Bl
`
`1801
`USER SELECTS TEST
`PARAMETERS
`1803
`DETERMINE UPPER
`PMTVOLTAGE
`1805
`SELECT MEDIUM AND
`LOW PMT VOLTAGES
`
`MEASURE PMT
`SIGNAL FOR HIGH
`REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`COEFFICIENT
`
`1815
`. - - - -~ -~ - ,
`READ SAMPLE WELL
`w WITH SINGLE FLASH
`
`YES
`
`1835
`~---'----~
`READ SAMPLE WELL w
`WITH PRESELECTED
`NUMBER OF FLASHES
`
`1837
`
`2003
`
`w=n+1
`
`2015
`2017
`. - - - - - ' - -
`
`PROCESS ~-
`
`DATA
`
`w=n+1
`
`2015
`
`PROCESS
`DATA
`
`2017
`
`FIG. 20.
`
`Agilent Exhibit 1240
`Page 16 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 16 of 26
`
`US 6,316,774 Bl
`
`1801
`
`2101
`
`2103
`
`1807
`
`1809
`
`2105
`
`210
`
`USER SELECTS TEST
`PARAMETERS
`
`LOW PMT VOLT AGE SET
`
`LOW GAIN SET
`
`MEASURE PMT SIGNAL
`FOR HIGH REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`COEFFICIENT
`
`w=n
`
`READ SAMPLE WELL w
`WITH SINGLE FLASH
`
`DETERMINE
`APPROPRIATE PMT
`VOLTAGE AND GAIN FOR
`SAMPLE WELL w
`
`2109 w=n+1
`STORE PMT VOLTAGE
`AND GAIN FOR SAMPLE
`WELLw
`
`NO
`
`w= n
`
`2113
`
`2115
`
`SET PMT VOLTAGE AND
`GAIN FOR SAMPLE WELL
`w PER PRE-DETERMINED
`VALUES
`
`READ SAMPLE WELL w
`WITH SELECTED
`NUMBER OF FLASHES
`
`2117
`
`w=n+1
`
`PROCESS DA TA
`
`FIG. 21.
`
`Agilent Exhibit 1240
`Page 17 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 17 of 26
`
`US 6,316,774 Bl
`
`USER SELECTS TEST
`PARAMETERS
`
`LOW PMT VOLTAGE SET
`
`LOW GAIN SET
`
`MEASURE PMT SIGNAL
`FOR HIGH REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`COEFFICIENT
`
`w= n
`
`READ SAMPLE WELL w
`WITH SINGLE FLASH
`
`DETERMINE
`APPROPRIATE PMT
`VOLTAGE AND GAIN FOR
`SAMPLE WELL w
`
`AND GAIN FOR SAMPLE
`WELLw
`
`1801
`
`2101
`
`2103
`
`1807
`
`1809
`
`2105
`
`2107
`
`w=n+1
`
`NO
`
`SET PMT VOLTAGE AND
`GAIN TO TYPE m
`
`READ SAMPLE WELL v
`WITH SELECTED
`NUMBER OF FLASHES
`
`ALL
`SAMPLE WELLS
`REQUIRING THESE PMT
`VOLTAGE AND GAIN
`SETTINGS
`
`2205
`
`v=z+1
`
`2213
`
`m = m + 1
`
`NO
`
`2211
`2215
`
`PROCESS DATA
`
`SORT PMT VOLTAGE AND
`GAIN FOR ALL SAMPLES
`
`2201
`
`FIG. 22.
`
`Agilent Exhibit 1240
`Page 18 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 18 of 26
`
`US 6,316,774 Bl
`
`2301
`
`2303
`
`1803
`
`1807
`
`1809
`
`2305
`
`USER SELECTS SPECTRUM MODE
`
`USER INPUTS REQUIRED TEST
`PARAMETERS
`
`DETERMINE UPPER PMT VOLTAGE
`
`MEASURE PMT SIGNAL FOR HIGH
`REFERENCE
`
`DETERMINE PMT CALIBRATION
`COEFFICIENT
`
`7\.=z
`
`w=n
`
`READ SAMPLE WELL w WITH
`SELECTED NUMBER OF FLASHES
`
`w=n+1
`
`2309
`
`z = z + INCREMENT 1-------'
`
`NO
`
`NO
`
`2313
`
`PROCESS DATA
`
`FIG. 23.
`
`Agilent Exhibit 1240
`Page 19 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 19 of 26
`
`US 6,316,774 Bl
`
`\\9
`
`\29
`
`240\
`
`242\
`
`\
`' 24\9
`\
`0 lz4z3
`
`- -
`
`FIG. 24.
`
`Agilent Exhibit 1240
`Page 20 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 20 of 26
`
`US 6,316,774 Bl
`
`! W 2409
`
`~~ ~
`
`I
`'
`
`2509
`
`1201,
`
`FIG. 25.
`
`Agilent Exhibit 1240
`Page 21 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 21 of 26
`
`US 6,316,774 Bl
`
`2409
`
`,-1201
`
`FIG. 26.
`
`.,,.-1201
`
`FIG. 27.
`
`Agilent Exhibit 1240
`Page 22 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 22 of 26
`
`US 6,316,774 Bl
`
`2413
`
`FIG. 28.
`
`125
`
`,,
`
`: '<' ~
`JI '-L \
`/
`~/,,I·~
`Exc1TAT10N ·<-v L 1 _J.
`REFLECTED
`··
`BEAM
`BEAM
`EMISSION
`BEAM
`FIG. 29.
`
`EMISSION
`BEAM
`r··r
`I t I
`\ I AREA OF DETECTED
`EMISSION
`
`EXCITATION,r---- - -
`BEAM L----- -/ \
`
`·--
`
`--- "l
`-----.J
`
`115
`
`\
`/
`I
`I
`•
`I
`J-. •. -J
`
`FIG.30
`
`Agilent Exhibit 1240
`Page 23 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 23 of 26
`
`US 6,316,774 Bl
`
`18000
`
`16000
`
`14000
`
`12000
`
`10000
`3105,
`8000
`
`6000
`
`4000
`
`2000
`
`~3103
`
`FIG. 31.
`
`Agilent Exhibit 1240
`Page 24 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 24 of 26
`
`US 6,316,774 Bl
`
`C")
`0
`N
`C")
`
`\_
`
`z
`0 -
`I-
`<(
`WN~
`.....J 0::: w
`0... w I-
`~ I- U)
`<( u >-
`(f) <( U)
`0:::
`<(
`I u
`
`a
`
`,,
`
`...-
`a
`N
`C")
`\_
`
`z
`0
`w I- ~
`.....J <( w
`0... 0::: I-
`~ <( U)
`<( 0... >-
`(/) w U)
`0:::
`0...
`
`CJ)
`0
`N
`C")
`
`\_
`
`~
`
`L.()
`0
`N
`C")
`
`\_
`
`~ .
`
`t'-
`0
`N
`C")
`
`\_
`
`>-
`0:::
`0
`~ w
`~
`
`1.
`
`0:::
`0
`(f)
`U)
`w
`u
`0
`0:::
`0...
`
`,I
`
`~
`u
`0
`.....J u
`
`...--
`N
`C")
`
`\_
`
`-
`.
`
`L.()
`...--
`N
`C")
`
`\_
`
`r
`
`...--
`...--
`N
`C")
`
`\_
`
`-
`
`r
`
`0:::
`w
`I-
`I-
`0
`.....J
`0...
`
`0:::
`0
`I-
`z
`0
`~
`
`0:::
`w
`I-
`z
`0:::
`Q_
`
`Agilent Exhibit 1240
`Page 25 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 25 of 26
`
`US 6,316,774 Bl
`
`INSERT SAMPLE PLATE
`
`PROGRAM SAMPLE PLATE
`CONFIGURATION
`
`PROGRAM COMPOSITIONS
`
`INITIATE FIRST SAMPLE
`PREPARATION AT TIME= x
`
`INITIATE SECOND SAMPLE
`PREPARATION AT TIME= x + L1
`
`INITIATE THIRD SAMPLE
`PREPARATION AT TIME= x + 2.6
`
`INITIATE FINAL SAMPLE
`PREPARATION AT TIME= x + z.6
`
`'
`'
`•
`•
`•
`'
`'
`'
`'
`'
`'
`
`1,,-- 3301
`
`v 3303
`
`I~ 3305
`
`~ 3307
`
`1.,,,-- 3309
`
`V 3311
`
`V 3313
`
`3315
`
`1.,,.--
`
`3317
`
`1.,,.--
`
`3319
`
`1.,,,--
`
`3321
`
`3323
`
`V
`
`REMOVE SAMPLE MICROPLATE FROM
`PREPARATION SYSTEM AND INSERT
`INTO CHARACTERIZATION SYSTEM
`
`INITIATE FIRST SAMPLE
`CHARACTERIZATION AT TIME= y
`
`INITIATE SECOND SAMPLE
`CHARACTERIZATION AT TIME= y + L1
`
`INITIATE THIRD SAMPLE
`CHARACTERIZATION AT TIME = y + 2L1
`
`INITIATE FINAL SAMPLE
`CHARACTERIZATION AT TIME= y + zL'.1
`FIG. 33.
`
`Agilent Exhibit 1240
`Page 26 of 42
`
`

`

`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 26 of 26
`
`US 6,316,774 Bl
`
`SAMPLE 1 PREPARATION AT ~ 3401
`TIME t 1
`
`SAMPLE PREPARATION AT TIME v- 3403
`
`I
`
`t2
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`SAMPLE CHARACTERIZATION AT v- 3405
`
`TIME tn
`
`FIG. 34.
`
`Agilent Exhibit 1240
`Page 27 of 42
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`US 6,316,774 Bl
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`1
`OPTICAL SYSTEM FOR A SCANNING
`FLUOROMETER
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`2
`fluorescence, thereby reducing the noise signal level in the
`detector and increasing the sensitivity of fluorescence detec(cid:173)
`tion.
`From the foregoing, it is apparent that a high sensitivity,
`5 wavelength scanning fluorometer is desired.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a method and apparatus
`for determining the fluorescence, luminescence, or light
`10 absorption of a sample. The sample may either be contained
`within a cuvette or within one or more sample wells of a
`multi-assay plate. The system is designed to accommodate a
`variety of different multi-assay plates in which the plate
`dimensions as well as the number of sample wells varies.
`In one aspect of the invention, an excitation means is
`provided for either fluorescence or absorption measure(cid:173)
`ments. The excitation means includes a broadband light
`source, a monochromator, and a series of optical filters. This
`combination of optical components allows the excitation
`20 wavelength to be tuned to a predetermined value within a
`relatively wide wavelength band. Depending upon the dis(cid:173)
`persion of the components, bandpass values of approxi(cid:173)
`mately 10 nanometers are commonly achievable. A similar
`optical configuration is used to detect the emissions from the
`25 sample (i.e., fluorescence or luminescence) or the amount of
`light absorbed by the sample. The detection means includes
`a photomultipler tube detector, a diffraction grating, and a
`series of optical filters.
`In another aspect of the invention, multiple optical fibers
`are coupled to the excitation source, thus allowing the
`system to be quickly converted from one optical configu(cid:173)
`ration to another. For example, the source can be used to
`illuminate either the top or the bottom of a sample well
`35 within a multi-assay plate or to illuminate a single cuvette
`cell. Similarly, multiple optical fibers are coupled to the
`detector. The multiple detector fibers allow the system to be
`easily converted from detecting fluorescence or lumines(cid:173)
`cence to detecting the amount of excitation light passing
`40 through the sample (i.e., for absorption measurements). The
`multiple detection fibers also allow the optical configuration
`to be converted to match the excitation configuration, e.g.,
`cuvette cell versus multi-assay plate.
`In another aspect of the invention, the excitation light and
`45 the detected sample emissions pass to and from an optical
`head assembly via a pair of optical fibers. The optical head
`assembly is coupled to a pair of guide rails and controlled by
`a step motor, thus allowing the head assembly to be driven
`along one axis of a multi-assay plate. The multi-assay plate
`50 is mounted to a carriage assembly that is also coupled to a
`pair of guide rails and controlled by a step motor. The
`carriage assembly drives the multi-assay plate along a
`second axis orthogonal to the first axis.
`In another aspect of the invention, the system is designed
`to accommodate a wide range of sample intensities
`automatically, such as would be expected from a group of
`random samples within a multi-assay plate. In order to
`accommodate varying intensities, a photomultiplier tube
`detector is used and the voltage is automatically varied in
`order to change its gain. The automatic voltage adjustment
`is performed in three steps, each providing a nominal
`dynamic range of three decades. Alternatively, the voltage
`adjustment can be performed in more than three steps
`employing finer gradations of dynamic range.
`In another aspect of the invention for use with a multi(cid:173)
`assay plate configuration, the system is designed to mini(cid:173)
`mize the effects of temperature drop from one sample to
`
`This application claims benefit from Provisional Appli(cid:173)
`cation Ser. No. 60/096,999, filed Aug. 18, 1998.
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to detection
`systems, and more particularly, to a method and apparatus
`for detecting fluorescence, luminescence, or absorption in a
`sample.
`
`BACKGROUND OF THE INVENTION
`
`15
`
`30
`
`In biology as well as other related scientific fields,
`samples are routinely characterized by examining the prop(cid:173)
`erties of fluorescence, luminescence, and absorption. Typi(cid:173)
`cally in a fluorescence study, selected tissues, chromosomes,
`or other structures are treated with a fluorescent probe or
`dye. The sample is then irradiated with light of a wavelength
`that causes the fluorescent material to emit light at a longer
`wavelength, thus allowing the treated structures to be iden(cid:173)
`tified and to some extent quantified. The wavelength shift
`between the peak excitation wavelength and the peak fluo(cid:173)
`rescence wavelength is defined as the Stokes shift and is the
`result of the energy losses in the dye molecule.
`In a luminescence study, the sample material in question
`is not irradiated in order to initiate light emission by the
`material. However, one or more reagents may have to be
`added to the material in order to initiate the luminescence
`phenomena. An instrument designed to monitor lumines(cid:173)
`cence must be capable of detecting minute light emissions,
`preferably at a predetermined wavelength, and distinguish(cid:173)
`ing these emissions from the background or ambient light.
`In a typical light absorption study, a dye-containing
`sample is irradiated by a light source of a specific wave(cid:173)
`length. The amount of light transmitted through the sample
`is measured relative to the amount of light transmitted
`through a reference sample without dye. In order to deter(cid:173)
`mine the concentration of dye in a sample, both the light
`absorption coefficient ( at the wavelength used) and the
`pathlength through the sample must be known. Other rela(cid:173)
`tive measurements may also be of interest, for example
`determining the wavelength dependence of the absorption.
`In general, an instrument designed to determine the fluo(cid:173)
`rescence of a sample requires at least one light source
`emitting at one or more excitation wavelengths and a
`detector for monitoring the fluorescence emissions. This
`same instrument can often be used for both luminescence
`and absorption measurements with only minor changes.
`U.S. Pat. No. 4,626,684 discloses a fluorescence mea(cid:173)
`surement system for use with a multi-assay plate. The 55
`disclosed system uses concave holographic gratings to con(cid:173)
`trol both the excitation and emission detection wavelengths.
`Optical fibers are used to couple the optical scanning head
`to both the source and detector subassemblies. The paths of
`both the excitation light and the fluorescent emissions are 60
`orthogonal to the surface of the material under study.
`U.S. Pat. No. 4,501,970 discloses a fluorometer for use
`with multi-assay plates. The disclosed system directs the
`excitation beam of light through the open top of the sample
`holding vessel and receives the fluorescent emission through 65
`this same opening. The system uses a series of mirrors and
`masks to decouple the excitation light from the emitted
`
`Agilent Exhibit 1240
`Page 28 of 42
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`US 6,316,774 Bl
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`5
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`3
`another that are due to evaporative cooling. Specifically, the
`plate holding carriage moves the multi-assay plate to a
`sample holding area between readings. Within the sample
`holding area the multi-assay plate is confined by an upper or
`lid surface that is close to the upper surface of the multi-
`assay plate. The sides of the multi-assay plate may also be
`confined. When the multi-assay plate is within this area the
`relative humidity above the plate rises to more than 90
`percent, thus reducing evaporative cooling. This aspect of
`the invention is preferably coupled to a temperature regu- 10
`lation and air circulation system.
`In another aspect of the invention, an optical scanning
`head assembly is used that includes mirrored optics for
`coupling an excitation source to the sample and the emitted
`light to a detector. An ellipsoidal focussing mirror is used to 15
`magnify and focus the source light projected from an optical
`fiber onto the sample. A portion of the source light is
`reflected by a beamsplitter onto a reference detector used to
`monitor the output of the source. The light from the ellip(cid:173)
`soidal mirror passes through an aperture in a second ellip- 20
`soidal mirror prior to impinging upon the sample. The light
`emitted by the sample within the sample well (e.g.,
`fluorescence) is reflected by the second ellipsoidal mirror
`and imaged onto the entrance aperture of an optical fiber
`coupled to the detector subassembly. The optical axes of 25
`both mirrors are slightly offset from the sample well normal.
`The offset minimizes the amount of light reflected from the
`meniscus of the sample or the bottom surface of the sample
`well that enters the detection subassembly.
`In another aspect of the invention, time tags are recorded 30
`for samples contained within a multi-assay plate. The time
`tags can be used to monitor compositional time dependent
`properties, for example those associated with a kinetic
`reaction. The time tags can also be used to insure that a
`comparison of individual samples within a multi-assay plate 35
`is accurate and is not biased by variations in the amount of
`time passing between the steps of sample preparation and
`sample characterization. In one mode of time tagging, a time
`tag is recorded for each critical preparation step and each
`critical characterization step for every sample of interest. In 40
`a second mode of time tagging, only a single time tag is
`recorded for the entire multi-assay plate for each critical step
`of either preparation or characterization. In this mode,
`however, every sample of the multi-assay plate is sequen(cid:173)
`tially prepared or characterized with a set interval passing 45
`between the preparation or characterization of successive
`samples.
`A further understanding of the nature and advantages of
`the present invention may be realized by reference to the 50
`remaining portions of the specification and the drawings.
`
`4
`FIG. 9 is a perspective view of an emission filter wheel
`assembly;
`FIG. 10 is an illustration of an emission filter wheel,
`including filters;
`FIG. 11 is an illustration of a multi-assay plate carriage
`assembly according to the invention;
`FIG. 12 is an illustration of a scanning optical stage
`assembly according to the invention;
`FIG. 13 is an illustration of the combined carriage and
`optical stage assemblies;
`FIG. 14 is an illustration of a portion of the temperature
`control system used with the present invention;
`FIG. 15 is an illustration of the underside of the base
`assembly of the preferred embodiment of the invention;
`FIG. 16 is a block diagram of the detection scheme in the
`preferred embodiment of the invention;
`FIG. 17 is a block diagram outlining the wavelength
`optimization procedure;
`FIG. 18 illustrates the algorithm used when a plate is read
`using the automatic mode of the invention;
`FIG. 19 illustrates an alternative approach to the tech(cid:173)
`nique shown in FIG. 18;
`FIG. 20 illustrates a variation of the method illustrated in
`FIG. 18;
`FIG. 21 illustrates another approach that may be utilized
`by the present invention;
`FIG. 22 illustrates a slight variation of the method shown
`in FIG. 21;
`FIG. 23 illustrates a spectrum mode of analysis for use
`with the invention;
`FIG. 24 schematically illustrates the well optics;
`FIG. 25 is an exploded view of an optical scanning head
`according to the preferred embodiment of the invention;
`FIG. 26 is a perspective upper view of the optical scan(cid:173)
`ning head shown in FIG. 25;
`FIG. 27 is a perspective lower view of the optical scan-
`ning head shown in FIGS. 25 and 26;
`FIG. 28 is a detailed view of the apertured detection
`mirror used in the preferred embodiment of the optical
`scanning head;
`FIG. 29 is an illustration of an alternative optical con(cid:173)
`figuration for use with a sample well;
`FIG. 30 is an illustration of an alternative optical con(cid:173)
`figuration for use with a cuvette cell;
`FIG. 31 illustrates the relationship between the position of
`the excitation light in the sample well with the amount of
`light reflected into the detector fiber;
`FIG. 32 is a schematic illustration of the principal com(cid:173)
`ponents of a time tagging system according to at least one
`55 embodiment of the invention;
`FIG. 33 illustrates the methodology associated with the
`embodiment shown in FIG. 32; and
`FIG. 34 illustrates the methodology associated with an
`alternative time tagging embodiment.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 schematically illustrates the detection system of
`the present invention;
`FIG. 2 is an illustration of the outer casing of one
`embodiment of the invention;
`FIG. 3 is an illustration of the combined optical subas(cid:173)
`semblies;
`FIG. 4 is a perspective view of an excitation filter wheel; 60
`FIG. 5 is a perspective view of an excitation filter wheel
`assembly;
`FIG. 6 is an exploded view of a shutter assembly;
`FIG. 7 is an illustration of the combined shutter plates 65
`utilized in the shutter assembly shown in FIG. 6;
`FIG. 8 is an illustration of a PMT housing and slit;
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`System Overview
`
`FIG. 1 schematically illustrates the principal components
`of at least one embodiment of a scanning fluorometer system
`100 according to the present invention. Preferably system
`
`Agilent Exhibit 1240
`Page 29 of 42
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`US 6,316,774 Bl
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`10
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`5
`100 is constructed utilizing subassembly modules. This
`module approach offers several benefits. First, it allows a
`non-functioning subassembly to be easily removed and
`replaced with a functioning subassembly, thereby minimiz(cid:173)
`ing the amount of time that the system is inoperable. Second, 5
`modules can be replaced or augmented as either the user's
`requirements change, or as improved subassemblies become
`available, thus providing for system growth. Third, this
`approach allows for increased on-site calibration and/or
`maintenance.
`A light source subassembly 101 within system 100 gen-
`erates illumination of a predetermined wavelength. Prefer(cid:173)
`ably the source for subassembly 101 is a broadband source,
`such as a xenon flash lamp 103. The light from lamp 103
`may pass through one or more apertures 105 in order to 15
`condition the light before passing through an optical filter
`107 mounted in an opening of a filter wheel 108. The
`wavelength of light emitted by source subassembly 101 is
`determined by a combination of filter 107, a movable grating
`109, and apertures formed by the input apertures of optical 20
`fibers 119.
`The light from source subassembly 101 is used to either
`illuminate a well 125 of a multi-assay plate 111 contained
`within a multi-assay plate chamber subassembly 113 or a
`cuvette 115 within a cuvette chamber subassembly 117.
`Multi-assay plate 111 is retained by a holding fixture. The
`light from source subassembly 101 is transmitted to multi(cid:173)
`assay plate chamber subassembly 113 or cuvette chamber
`subassembly 117 via a selected fiber of optical fibers 119.
`Furthermore, the source light can be transmitted either
`through the open top portions 124 of wells 125 or through
`transparent closed bottom portions 126 of wells 125, the
`selection of which is determined by the particular optical
`fiber 119 selected to couple source subassembly 101 to
`multi-assay plate 111. An optical shutter 121 within source
`subassembly 101 establishes which of fibers 119 receives
`light from source 103. One or more focussing mirrors 123
`focus the light passing through fibers 119 into the chamber
`of interest, i.e., multi-assay plate well 125 or cuvette 115.
`The light, either from cuvette 115, top portion 124 of well
`125, or bottom portion 126 of well 125, is collected with
`optics 127. The collected light can be either light emitted as
`fluorescence or luminescence, or transmitted light used for
`an absorption measurement. The collected light passes
`through a selected optical fiber of fibers 129 to a detection
`subassembly 131. When transmitted light is used for an
`absorption measurement in wells 125, the preferred configu(cid:173)
`ration is to pass the light first through top portion 124 of
`wells 125, then through the sample materials contained
`within wells 125, and finally through the bottom portion 126
`of wells 125. During absorption measurements, the trans(cid:173)
`mitted light is collected by optics 127 positioned under
`multi-assay plate 111. The collected light is then focused
`onto a selected fiber 129 for transmission to detector 135 in
`detector subassembly 131. In a first alternative configuration
`used for absorption measurements, as in the above configu(cid:173)
`ration the light enters well 125 through top portion 124.
`After the light passes through the sample materials within
`well 125, however, it is reflected back by a mirror under(cid:173)
`neath of the sample well (not shown) and collected by optics
`positioned above the well (not shown). In a second alterna(cid:173)
`tive configuration a detector, preferably a photodiode, is
`located directly under the well (not shown) and collects the
`light transmitted through well 125 and the sample materials
`contained therein. In a third alternative configuration (not
`shown) the light enters well 125 through bottom portion 126,
`passes through the sample materials, passes through top
`
`6
`portion 124, and is then collected and focussed onto a
`detector. In this configuration the detector may either be
`mounted remotely or be mounted in close proximity to top
`portion 124.
`A shutter 133 determines which fiber 129 is monitored by
`subassembly 131. The light from a selected fiber 129 is
`focussed onto a detector 135 by a movable, focussing
`grating 137. Preferably detector 135 is a photomultiplier
`tube (i.e., PMT). The light may pass through one or more
`apertures 141 to reduce stray light before impinging on
`detector 135. The combination of grating 137, aperture 141,
`and a filter 139 mounted in an opening of a filter wheel 140
`determines the wavelength oflight detected by detector 135.
`Grating 109 allows the excitation wavelength to be con-
`tinuously varied over a relatively wide wavelength band.
`Similarly, grating 137 allows the detection wavelength to be
`continuously varied over a wide range of wavelengths. In the
`preferred embodiment of the invention, gratings 109 and
`137 each have a focal length of approximately 100
`millimeters, thus allowing excitation subassembly 101 and
`detection subassembly 131 to be relatively compact. As the
`gratings are preferably holographic gratings with 1200
`grooves per millimeter, the dispersion of the gratings with
`this focal length provides a nominal 10 nanometer bandpass.
`25 In a preferred embodiment of the invention, the blaze angle
`of the gratings is 500 nanometers. However, the gratings
`may be blazed at different angles, thus further enhancing the
`decoupling of the excitation and fluorescence wavelengths.
`Preferably the arc of lamp source 103 is focused onto the
`entrance aperture of fiber 119.
`FIG. 2 illustrates the outer casing of one embodiment of
`the invention. In this embodiment a multi-assay plate 111
`that is ready for testing is placed within reading chamber 202
`of instrument 203 via a housing door 205. In at least one
`embodiment of the invention, the instrument can also be
`used to test a cuvette, preferably by inserting the cuvette into
`a cuvette port 209. A control panel 211 provides a user
`interface, allowing the user to initiate testing as well as set
`various testing protocols. Preferably control panel 211 also
`includes a simple r