(12) United States Patent
`Giebeler et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,316,774 B1
`Nov. 13, 2001
`
`US006316774Bl
`
`(54) OPTICAL SYSTEM FORA SCANNING
`FLUOROMETER
`
`W0 97/1352
`
`3/1997 (W0).
`
`(75)
`
`Inventors: Robert Giebeler, San Jose; David G.
`Ogle, Los Altos; Roger Kaye,
`M01lI1taiI1 VieW, all Of CA (US)
`_
`_
`.
`(73) Assignee: Molecular Devices Corporation,
`S”HHyVa1°> CA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) APP1. Nu; 09/274,753
`
`(22)
`
`Filed:
`
`Mar. 23, 1999
`
`Related US. Application Data
`Provisional application No. 60/096999 filed on Aug. 18.
`1998.
`’
`’
`'
`7
`
`..................................................... G01N’21/64
`.....................................
`
`Int.Cl.
`U.S. Cl.
`_
`Fleld Of Search ..............................
`
`250/4611
`
`2’ 0/4591’ 4612
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,401,573 *
`7/1984 Lucht et al.
`...................... 250/458.1
`4,501,970
`2/1985 Nelson .
`4,525,584
`12/1985 panda ,
`..................... .. 250/458.1
`4,691,110 *
`9/1987 Nebe et al.
`4,945,250 *
`7/1990 B0We11 et a1~
`~~~~~~~~~~~~~~~~~~ ~~ 250/4611
`5,133,936 *
`7/1992 Umetsu et al.
`...................... .. 422/64
`51591981
`1/1997 He1e1fingere1a1~~
`5,784,152
`7/1998 Heffelfingeretal.
`6,042,785 *
`3/2000 Harju ................................... .. 422/52
`FOREIGN PATENT DOCUMENTS
`
`(60)
`
`(51)
`
`(56)
`
`* cited by examiner
`
`Primary Examiner—Constantine Hannaher
`Assistant Examiner—Otilia Gabor
`(74) Attorney) Agent) 0,. Fl-,,m_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 tuiiethe excitation
`Wavelength to a predetermmed Value Wlthm a relanvely
`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
`and focus the
`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
`apemm’ in " Second. "1hpti“.‘1 mirror prior to 1111P111g.111$11P°11
`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
`1' htl
`ff tf
`th
`1
`'11
`1. Th
`'
`iflfsiilgmmfilziitLZmamZ§§f‘1§§1?‘L‘§§l?§ed ffofltrffii
`.
`.1
`g
`_
`_
`111e111Se11S 01 the Sample 01 the h0tt0II1 Surtaee 01 the Sample
`well that enters the detection subassembly.
`
`WO 97/11351
`
`3/1997 (W0) .
`
`32 Claims, 26 Drawing Sheets
`
`
`
`fur
`
`H5
`
`‘E7
`
`l
`
`I23
`H3
`
`/-I29
`
`I27
`I24
`
`ll
`
`I25
`
`‘35
`
`“III
`
`I29
`
`/
`
`:27
`
`|lZ5
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 1 of 26
`
`US 6,316,774 B1
`
`F/GI
`
`/.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`S”U
`
`eM
`
`m
`
`7
`
`Pm
`
`2tUnuan
`
`MF2W3!n1m...V.n0F-Nn,2.E
`
`m.
`
`m2
`
`M.TfBM2
`
`12B.o.Mx
`WE
`
`6HWIB.11.F
`
`OW%
`
`0
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 3 of 26
`
`US 6,316,774 B1
`
`FIG? 3.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 4 of 26
`
`US 6,316,774 B1
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13,2001
`
`Sheet 5 of 26
`
`US 6,316,774 B1
`
`FIG? 8.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 6 of 26
`
`US 6,316,774 B1
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`tHetaP&U
`
`1002’BWN
`
`cl0
`
`1
`
`7OmN%I
`
`mF
`
`m.m
`
`B.o.Mx.mMmwWS
`
`HOmE
`
`m
`
`THERMO FISHER EX. 1020
`
`

`
`tnetaPS_U
`
`m.N
`
`mNm,
`
`08
`
`6
`
`wP.%.
`
`0.71.T
`
`MF
`
`12B.o.Mx.wMmESW.UI
`
`
`
` F0W%
`
`0
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 9 of 26
`
`US 6,316,774 B1
`
`FIG /4.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`tHEtaPS“U
`
`m.N
`
`mmm,
`
`m
`
`sa
`
`m.b.
`
`H
`
`6E2HcmT
`
`12B.o.Mx.mMmESW.UI
`
`F0W
`
`0
`
`THERMO FISHER EX. 1020
`
`

`
`U
`
`tnCtaP
`
`002’3
`
`MS
`
`62M
`
`3
`
`7,6
`
`m
`
`S_Sm:
`
`ézwa
`
`9:
`
`
`
`M0.25:2:Eoo_Emommmoofi935&8>1
`
`
`
`
`
`a_2<m_EMExm:n__::s_So:moZm8E_E:Mz_<owm:n_2<m
`
`
`
`M.m:..0?‘
`
`
`
`
`
`
`
`
`
`
`
`
`mobmmamomaom6,m_o<_mm<o1mm_:msmmm<ENE:$250wflab:4<o_Eowmmmmmfibfimazzo_fiw__:¢%<omozzomzoozozmoE2o~_:oozos_moz_zz<om
`
`
`
`
`
`
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 12 of 26
`
`US 6,316,774 B1
`
`SELECT EMISSION
`
`WAVELENGTH
`
`SCAN EXCITATION
`
`WAVELENGTH
`
`
`
`
`1701
`
`1703
`
`
`
`
`DETERMINE PEAK
`
`1705
`
`EXCITATION WAVELENGTH
`
`FIX EXCITATION
`
`1707
`
`WAVELENGTH AT PEAK
`
`SCAN EMISSION
`
`WAVELENGTH
`
`1709
`
`DETERMINE PEAK EMISSION
`
`1710
`
`\NAVELENGTH
`
`SELECT EMISSION FILTER
`
`FIx EMISSION FILTER
`
`1711
`
`1712
`
`1713
`
`FIX EMISSION WAVELENGTH
`
`READ SAMPLES BY END
`
`1715
`
`POINT OR KINETIC MODE
`
`
`
`FIG. 17.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13,2001
`
`Sheet 13 of 26
`
`US 6,316,774 B1
`
`1801
`USER SELECTS TEST
`
`13/26
`
`MEASURE PMT
`
`1803
`
`SIGNAL FOR HIGH
`
`PMT VOLTAGE
`1305
`
`LOW PMT VOLTAGES
`
`DETERMINE PMT
`
`CALIBRATION
`
`COEFFICIENT
`
`1807
`
`1809
`
`1811
` MORE
`THAN 1 FLASH
`
`YES
`
`1813
`
`
`
`
`YES TAG WELL
`
`LOCAHON
`
`
`
`READ SAMPLE WELL W
`WITH PRESELECTED
`NUMBER OF FLASHES
`
`AVERAGE DATA
`
`1835
`
`1837
`
`1819
`
`
`
`SATURATED?
`
`
`
`
`1827
`
`NO
`
`PROCESS
`DATA
`
`1829
`
`1333
`
`NO PROCESS
`T
`DA A
`
`1831
`
`DECREMENT FIVIT
`VOLTAGE
`
`1833
`
`PROCESS NO
`DATA
`
`YES
`
`1831
`
`DECREMENT PMT
`
`FIG 18_
`
`VOLTAGE
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 14 of 26
`
`US 6,316,774 B1
`
`USER SELECTS TEST
`PARAMETERS
`
`1801
`
`USER SELECTS PMT
`VOLTAGE
`
`MEASURE FMT SIGNAL
`FOR HIGH REFERENCE
`
`DETERMINE PMT
`CALIBRATION
`
`COEFFICIENT
`
`1901
`
`1807
`
`1809
`
`1811
` MORE
`THAN 1 FLASH
`
`YES
`
`NO
`1813
`
`1313
`
`W = n
`
`W : n
`
`
`
`1835
`READ SAMPLE WELL
`
`w WITH
`
`PRESELECTED
`
`
`
`
`NUMBER OF FLASHES
`
`
`Y
`
`ES
`
`1903
`
`PROCESS DATA
`
`ES
`
`Y
`
`1903
`
`PROCESS DATA
`
`FIG. 19.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 15 of 26
`
`US 6,316,774 B1
`
`1807
`
`
`
`
`
`MEASURE pm
`
`
`
`SIGNAL FOR HIGH
`REFERENCE
`
`
`
`
`
`
`
`DETERMINE PMT
`CALIBRATION
`COEFF|C|ENT
`
`
`
`
`
`181 1
`
`1801
`USER SELECTS TEST
`
`1803
`DETERMINE UPPER
`
`1805
`SELECT MEDIUM AND
`LOW PMT VOLTAGES
`
`NO
`1813
`
`THAN 1 FLASH
`SELECTED?
`
`
`YES
`
`1813
`
`W = n
`
`1835
`
`1837
`
`READ SAMPLE WELL w
`
`WITH PRESELECTED
`
`NUMBER OF FLASHES
`
`
`
`NO
`
`1816
`PMT
`SATURATED?
`
`
`
`
`PMT VOLTAGES
`AVAILABLE?
`
`2005
`
`
`
`PMT VOLTAGES
`AVAKABLE?
`
`2017
`
`2015
`
`2017
`
`PROCESS
`DATA
`
`FIG. 20.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 16 of 26
`
`US 6,316,774 B1
`
`2115
`
`SET PMT VOLTAGE AND
`
`
`GAIN FOR SAMPLE WELL
`
`
`
`
`W PER PRE-DETERMINED
`
`VALUES
`
`
`
`READ SAMPLE WELL w
`
`
`
`
`
`WITH SELECTED
`
`NUMBER OF FLASHES
`
`
`
`w=n+1
`
`ALL
`
`
`
`
`NO
`TESTED?
`
`YES
`2119
`2121
`
`USER SELECTS TEST
`PARAMETERS
`
`LOW PMT VOLTAGE SET
`
`LOW GAIN SET
`
`MEASURE PMT SIGNAL
`FOR HIGH REFERENCE
`
`1801
`
`21 01
`
`2103
`
`1807
`
`CALIBRATION
`COEFFICIENT
`
`W = “
`
`PROCESS DATA
`
`READ SAMPLE WELL w
`WITH SINGLE FLASH
`
`DETERMINE
`
`
`
`
`APPROPRIATE PMT
`
`VOLTAGE AND GAIN FOR
`SAMPLE WELL w
`
`STORE PMT VOLTAGE
`
`AND GAIN FOR SAMPLE
`
`WELL w
`
`
`
`ALL
`
`
`
`
`SAMPLE WELLS
`
`TESTED?
`
`2113
`
`FIG. 21.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 17 of 26
`
`US 6,316,774 B1
`
`2203
`
`V = Z
`
`SET PMT VOLTAGE AND
`GAIN TO TYPE m
`
`READ SAMPLE WELL v
`
`WITH SELECTED
`NUMBER OF I.-LASHES
`
`
`
`
`
`
`
`REQUIRING THESE PMT
`VOLTAGE AND GAIN
`SETTINGS
`
`PROCESS DATA
`
`USER SELECTS TEST
`PARAMETERS
`
`LOW PMT VOLTAGE SET
`
`1801
`
`2101
`
`LOW GAIN SET
`
`MEASURE PMT SIGNAL
`FOR HIGH REFERENCE
`
`1807
`
`DETERMINE PMT
`CALIBRATION
`
`COEFFICIENT
`
`W = H
`
`1809
`
`2105
`
`READ SAMPLE WELL w
`WITH SINGLE FLASH
`
`
`
`
`
`APPROPRIATE PMT
`VOLTAGE AND GAIN FOR
`SAMPLE WELL w
`
`DETERMINE
`
`w=n+I
`
`STORE PMT VOLTAGE
`AND GAIN FOR SAMPLE
`WELL w
`
`
`
`ALL
`
`
`
`
`SAMPLE WELLS
`TESTED’?
`
`SORT PMT VOLTAGE AND
`GAIN FOR ALL SAMPLES
`
`2201
`
`FIG. 22.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 18 of 26
`
`US 6,316,774 B1
`
`USER SELECTS SPECTRUM MODE
`
`USER INPUTS REQUIRED TEST
`PARAMETERS
`
`DETERMINE UPPER PMT VOLTAGE /-
`
`230‘!
`
`2303
`
`1803
`
`MEASURE PMT SIGNAL FOR HIGH /4307
`REFERENCE
`
`DETERMINE PMT CALIBRATION
`COEFFICIENT
`
`/1809
`
`READ SAMPLE WELL w WITH
`
`SELECTED NUMBER OF FLASHES
`
`
`
`
`
`ALL SAMPLE WELLS TESTED?
`
`
`
`
`2307
`
`YES
`
`2309
`
`NO
`
`z=z+lNCREMENT
`
`2311
`
`YES
`
`2313
`
`PROCESSDATA
`
`FIG. 23.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 20 of 26
`
`US 6,316,774 B1
`
`FIG 25.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`m.N
`
`0Nm,
`
`0
`
`1F
`
`m7.w2%G.
`
`I
`
`m.%fT
`
`F0W
`
`0
`
`12B.o.Mx.wMmESH
`
`SUI
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 22 of 26
`
`US 6,316,774 B1
`
`24l3
`
`24H
`
`FIG 28.
`
`
`/./J’ K;
`EXC|TATION‘\/L \\
`BEAM
`--
`EMISSION
`BEAM
`
`/
`
`:‘<’:
`
`REFLECTED
`BEAM
`
`F/(:? 2.9
`
`EMISSION
`
`'\
`
`AREA OF DETECTED
`
`EMISSION
`
`---#7
`-——-- __'s
`
`
`
`ExcnTAT1oN,r:_--~ —
`BEAM
`L_..-———— —
`
`H5
`
`\‘
`
`am;
`
`F/6.30
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 23 of 26
`
`US 6,316,774 B1
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`aPS”U
`
`m
`
`B
`
`MMH.m
`
`m8%8%m,8%
`
`w8%3%
`
`
`
`W_>_m+m>msm:m>m
`
`
`
`zo_»<N_wm_5<m<:ozo:<m<nm_mn_
`
`
`
`m#__2<mm:n_s_<m
`
`
`
`
`
`>mosm_>_mommmoomagood
`
`mRmmam:mmW.%m
`
`
`
`
`
` mM4.~....GE1.WXmMWSmmtofimo:zo_>_mmFz_EH
`
`0M
`
`0
`
`THERMO FISHER EX. 1020
`
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 25 of 26
`
`US 6,316,774 B1
`
`INSERT SAMPLE PLATE
`
`PROGRAM SAMPLE PLATE
`CONFIGURATION
`
`PROGRAM COMPOSITIONS
`
`INITIATE FIRST SAMPLE
`PREPARATION AT TIME = x
`
`INITIATE SECOND SAMPLE
`PREPARATION AT TIME = x + A
`
`3301
`
`3303
`
`3305
`
`3307
`
`3309
`
`INITIATE THIRD SAMPLE
`
`33“
`
`PREPARATION AT TIME = x + 2A
`
`INITIATE FINAL SAMPLE
`PREPARATION AT TIME = x + ZA
`
`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 + A
`
`INITIATE THIRD SAMPLE
`CHARACTERIZATION AT TIME = y + 241
`
`INITIATE FINAL SAMPLE
`CHARACTERIZATION AT TIME = y + 2A
`
`FIG. 33.
`
`3313
`
`3315
`
`3317
`
`3319
`
`3321
`
`3323
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`U.S. Patent
`
`Nov. 13, 2001
`
`Sheet 26 of 26
`
`US 6,316,774 B1
`
`SAMPLE 1 PREPARATION AT
`
`TIME t1
`
`SAMPLE PREPARATION AT TIME
`
`t2
`
`
`
`SAMPLE CHARACTERIZATION AT
`
`TIME tn
`
`3401
`
`3403
`
`3405
`
`
`
`
`
`FIG. 34.
`
`THERMO FISHER EX. 1020
`
`THERMO FISHER EX. 1020
`
`

`
`US 6,316,774 Bl
`
`1
`OPTICAL SYSTEM FOR A SCANNING
`FLUOROMETER
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`This application claims benefit from Provisional Appli-
`cation Ser. No. 60/096,999, filed Aug. 18, 1998.
`
`FIELD OF THE INVENTION
`
`invention relates generally to detection
`The present
`systems, and more particularly, to a method and apparatus
`for detecting fluorescence, luminescence, or absorption in a
`sample.
`
`BACKGROUND OF THE INVENTION
`
`In biology as well as other related scientific fields,
`samples are routinely characterized by examining the prop-
`erties of fluorescence, luminescence, and absorption. Typi-
`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-
`tificd and to some cxtcnt quantificd. The wavelength shift
`between the peak excitation wavelength and the peak fluo-
`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-
`cence must be capable of detecting minute light emissions,
`preferably at a predetermined wavelength, and distinguish-
`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-
`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-
`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-
`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-
`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-
`surement system for use with a multi-assay plate. The
`disclosed system uses concave holographic gratings to con-
`trol both the excitation and emission detection wavelengths.
`Optical fibcrs 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
`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
`this same opening. The system uses a series of mirrors and
`masks to decouple the excitation light from the emitted
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`fluorescence, thereby reducing the noise signal level in the
`detector and increasing the sensitivity of fluorescence detec-
`tion.
`
`From the foregoing, it is apparent that a high sensitivity,
`wavelength scanning fluorometer is desired.
`SUMMARY OF THE INVENTION
`
`The present invention provides a method and apparatus
`for determining the fluorescence,
`luminescence, or light
`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-
`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
`wavelength to be tuned to a predetermined value within a
`relatively wide wavelength band. Depending upon the dis-
`persion of the components, bandpass values of approxi-
`mately 10 nanometers are commonly achievable. A similar
`optical configuration is used to detect the emissions from the
`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-
`ration to another. For example, the source can be used to
`illuminate either the top or the bottom of a sample well
`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-
`cence to detecting the amount of excitation light passing
`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
`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
`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
`cmploying fincr gradations of dynamic range.
`In another aspect of the invention for use with a multi-
`assay plate configuration, the system is designed to mini-
`mize the effects of temperature drop from one sample to
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`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-
`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
`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-
`soidal mirror passes through an aperture in a second ellip-
`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
`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
`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
`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
`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-
`tially prepared or characterized with a set interval passing
`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
`remaining portions of the specification and the drawings.
`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-
`semblies;
`FIG. 4 is a perspective view of an excitation filter wheel;
`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
`utilized in the shutter assembly shown in FIG. 6;
`FIG. 8 is an illustration of a PMT housing and slit;
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`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-
`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-
`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-
`figuration for use with a sample well;
`FIG. 30 is an illustration of an alternative optical con-
`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 coin-
`ponents of a time tagging system according to at least one
`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.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`System Overview
`
`FIG. 1 schematically illustrates the principal components
`of at least one embodiment of a scanning fiuorometer system
`100 according to the present invention. Preferably system
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`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-
`ing the amount of time that the system is inoperable. Second,
`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-
`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
`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
`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-
`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-
`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-
`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-
`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-
`neath of the sample well (not shown) and collected by optics
`positioned above the well (not shown). In a second alterna-
`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
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`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 of light 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.
`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 readout system such as a LCD readout,
`thus providing the user with positive indications of selec-
`tions as well as status.
`
`Instrument 203 is preferably coupled to a data processing
`system 213 via a cable 215. Data processing system 213 is
`used to manipulate the data, store the data, and present the
`data to the user via either a monitor 217 or a printer 219.
`Depending upon the system configuration, processing sys-
`tem 213 can also be used to control the test itself (i.e., test
`initiation,
`test protocol settings, etc.).
`In the preferred
`embodiment, an internal processor controls at least the basic
`test parameters by controlling movable gratings 109 and
`137, filter wheels 108 and 140, shutters 121 and 133, and the
`relative movement of multi-assay plate 111 to excitation
`optics 123 and detection optics 127.
`FIG. 3 is an illustration of the optical subassemblies of the
`preferred embodiment of the invention. In order to decrease
`the overall size of system 100, in this embodiment of the
`invention both source subassembly 101 and detection sub-
`assembly 131 are contained on a single optical bench 301.
`Source 103 is mounted within a bracket 303 and coupled
`to a source high voltage power supply 305. Preferably
`source 103 is a xenon flash lamp due to its relatively wide
`emittance wavelength band, ranging from the ultraviolet to
`the infrared. In alternative embodiments, source 103 can be
`a mercury arc lamp, a laser, an incandescent lamp (e.g., a
`tungsten lamp), or other source. The light from source 103
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`passes through a filter wl1eel 108 containing a plurality of
`optical filters 107. Filters 107 can be bandpass filters (i.e.,
`pass a band of wavelengths), cutoff filters (i.e., only pass
`wavelengths above or below a predetermined wavelength),
`or any other type of optical filter that can be used to control
`the wavelength of light passing through the filter and
`impinging on optical grating 109. The position of filter
`wheel 108, and therefore the selected filter in the excitation
`beam path, is controlled by motor 309. Aposition sensor 311
`(e.g., an optical switch) or other means is used to determine
`the position of wheel 108, and thus the filter 107 within the
`beam path. Preferably motor 309 and position sensor 311 are
`coupled to a controller internal to the instrument.
`The light passing through the selected filter 107 in filter
`wheel 108 is rellected off of grating 109. Grating 109 is
`cou