(12) United States Patent
`Stumbo et al.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US006310687Bl
`US 6,310,687 Bl
`Oct. 30, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) LIGHT DETECTION DEVICE WITH MEANS
`FOR TRACKING SAMPLE SITES
`
`(75)
`
`(73)
`
`Inventors: David P. Stumbo, Belmont; Douglas
`N. Modlin, Palo Alto, both of CA (US)
`
`Assignee: LJL Biosystems, Inc., 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 0 days.
`
`(21) Appl. No.: 09/768,765
`
`(22)
`
`Filed:
`
`Jan. 23, 2001
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`Continuation of application No. PCT/USOO/18547, filed on
`Jul. 7, 2000.
`Provisional application No. 60/142,721, filed on Jul. 7,
`1999.
`
`(51)
`
`Int. CI?
`
`G01N 21/64
`
`(52) U.S. Cl.
`
`356/317; 356/417; 250/458.1;
`422/63; 422/82.08
`356/317, 318,
`Field of Search
`356/417; 250/458.1, 459.1, 461.1, 461.2;
`422/63, 65, 82.05, 82.08
`
`(58)
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,645,800 * 7/1997 Masterson et al.
`5,888,454 * 3/1999 Leistner et al.
`* cited by examiner
`Primary Examiner-F. L. Evans
`(74) Attorney, Agent, or Firm-Kolisch Hartwell Dickinson
`McCormack & Heuser
`(57)
`
`ABSTRACT
`
`422/65
`422/65
`
`Apparatus and methods for optical detection with improved
`read speed and/or signal-to-noise ratio. These apparatus and
`methods may involve among others moving an sample
`substrate (108) while simulataneously detecting light trans(cid:173)
`mitted from one or more sample sites (110) on the substrate
`(108) by sequentially tracking the sample sites (110) as they
`move. A stage (101), movable in a first direction, supports
`the substrate (108). A detector (118) detects light emanating
`from an examination region (102) delimited by a detection
`initiation position (106a) and a detection termination posi(cid:173)
`tion (106b). An optical relay structure (122) transmit light
`from the examination region (102) to the detector (118). A
`scanning mechanism (120) simultaneously moves the opti(cid:173)
`cal relay structure (122) and the substrate in the first direc(cid:173)
`tion. The optical relay structure (122) tracks the substrate
`(108) between the detection initiation position (106a) and
`the detection termination position (106b).
`
`34 Claims, 10 Drawing Sheets
`
`~100
`
`116
`
`I
`
`/
`
`SUBSTRATE
`MOTION
`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 1 of 10
`
`US 6,310,687 Bl
`
`Fig. 1
`
`,r-100
`
`116
`
`Fig. 2
`
`~116
`
`~MOTION""" ~
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`I
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`
`MOTION
`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 2 of 10
`
`US 6,310,687 Bl
`
`Fig. 3
`
`204
`
`(
`
`~
`
`GALVO OR
`21
`~ ROTATING
`l"- f- POLYGON
`
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`./1
`
`~200
`
`COLLIMATED LIGHT BEAM:
`INPUT AND OUTPUT
`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 3 of 10
`
`US 6,310,687 Bl
`
`Fig. 4
`
`MOTION
`
`O LIGHT
`
`SOURCE
`
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`
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`
`1\
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`MIRRORED
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`
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`EMBODIMENT
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`
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`............0 DETECTOR
`
`,."
`,."
`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Fig.5A
`
`Oct. 30, 2001
`
`Sheet 4 of 10
`
`US 6,310,687 Bl
`
`CAPERTURE PLATE
`
`L.....-_--=:.....-.=...--=~.=..._.....I SUBSTRATE
`
`!
`
`LIGHT
`SOURCE
`
`I
`
`U
`
`U
`
`U
`
`I SUBSTRATE
`
`MOTION •
`SECOND ELLIPSE IS OPTIONAL, CAN
`ALSO WORK WITH 1 NON-DICHROIC
`
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`Fig. 5B
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`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 5 of 10
`
`US 6,310,687 Bl
`
`Fig.6A
`
`OPTICS AS
`IN FIG. SA
`
`-----l\-----APERTURE PLATE (FIXED)
`I
`\
`
`\
`
`I
`
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`
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`
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`
`LENS ROTATES
`ABOUT THIS POINT
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`LENS ROTATES
`ABOUT THIS POINT
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`APERTURE,
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`OPTICS AS
`IN FIG. SA
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`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 6 of 10
`
`US 6,310,687 Bl
`
`Fig. 7
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`CAN ALSO BE DONE WITH
`ELLIPSOIDAL MIRROR
`
`THERMO FISHER EX. 1032
`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 7 of 10
`
`US 6,310,687 Bl
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`
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`
`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 9 of 10
`
`US 6,310,687 Bl
`
`Fig. 10
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`

`
`u.s. Patent
`
`Oct. 30, 2001
`
`Sheet 10 of 10
`
`US 6,310,687 Bl
`
`Fig. 11
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`

`
`US 6,310,687 Bl
`
`5
`
`10
`
`1
`LIGHT DETECTION DEVICE WITH MEANS
`FOR TRACKING SAMPLE SITES
`CROSS-REFERENCE
`This application is a continuation of PCT Patent Appli-
`cation Ser. No. PCT/USOO/18547, filed Jul. 7, 2000, which
`is incorporated herein by reference.
`This application is based upon and claims priority from
`U.S. Provisional Patent Application Ser. No. 60/142,721,
`filed Jul. 7, 1999, which is hereby incorporated by reference.
`This application incorporates by reference the following
`U.S. patent application Ser. No. 08/840,553, filed Apr. 14,
`1997; Ser. No. 08/929,095, filed Sep. 15, 1997, now aban(cid:173)
`doned; Ser. No. 09/118,141, filed Jul. 16, 1998; Ser. No.
`09/144,575, filed Aug. 31, 1998, now U.S. Pat. No. 6,159,
`425; Ser. No. 09/144,578, filed Aug. 31, 1998; Ser. No. 15
`09/146,081, filed Sep. 2, 1998, now U.S. Pat. No. 6,187,267;
`Ser. No. 09/156,318, filed Sep. 18, 1998; Ser. No. 09/160,
`533, filed Sep. 24, 1998, now U.S. Pat. No. 6,097,025; Ser.
`No. 09/302,158, filed Apr. 29, 1999; Ser. No. 09/349,733,
`filed Jul. 8, 1999; Ser. No. 09/468,440, filed Dec. 21, 1999; 20
`Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/494,407,
`filed Jan. 28, 2000; Ser. No. 09/556,030, filed Apr. 20, 2000;
`and Ser. No. 09/596,444, filed Jun. 19, 2000.
`This application also incorporates by reference the fol(cid:173)
`lowing PCT patent application Ser. No. PCT/US99/01656, 25
`filed Jan. 25, 1999; Ser. No. PCT/US99/03678, filed Feb. 19,
`1999; Ser. No. PCT/US99/0841O, filed Apr. 16, 1999; Ser.
`No. PCT/US99/16057, filed Jul. 15, 1999; Ser. No. PCT/
`US99/16453, filed Jul. 21, 1999; Ser. No. PCT/US99/16621,
`filed Jul. 23, 1999; Ser. No. PCT/US99/16286, filed Jul. 26, 30
`1999; Ser. No. PCT/US99/16287, filed Jul. 26, 1999; Ser.
`No. PCT/US99/24707, filed Oct. 19, 1999; Ser. No. PCT/
`USOO/00895, filed Jan. 14, 2000; Ser. No. PCT/USOO/
`03589, filed Feb. 11,2000; Ser. No. PCT/USOO/04543, filed
`Feb. 22, 2000; Ser. No. PCT/USOO/06841, filed Mar. 15,
`2000; Ser. No. PCT/USOO/12277, filed May 3, 2000; Ser. 35
`No. PCT/USOO/15774, filed Jun. 9, 2000; Ser. No. PCT/
`USOO/16012, filed Jun. 9, 2000; and Ser. No. PCT/USOO/
`16025, filed Jun. 9, 2000.
`This application also incorporates by reference the fol(cid:173)
`lowing U.S. provisional patent application Ser. No. 60/143,
`185, filed Jul. 9, 1999; Ser. No. 60/153,251, filed Sep. 10,
`1999; Ser. No. 60/164,633, filed Nov. 10, 1999; Ser. No.
`60/165,813, filed Nov. 16, 1999; Ser. No. 60/167,301, filed
`Nov. 24, 1999; Ser. No. 60/167,463, filed Nov. 24, 1999; Ser.
`No. 60/178,026, filed Jan. 26, 2000; Ser. No. 60/182,036, 45
`filed Feb. 11,2000; Ser. No. 60/182,419, filed Feb. 14,2000;
`Ser. No. 60/184,719, filed Feb. 24, 2000; Ser. No. 60/184,
`924, filed Feb. 25, 2000; Ser. No. 60/190,265, filed Mar. 17,
`2000; Ser. No. 60/191,890, filed Mar. 23, 2000; Ser. No.
`60/193,586, filed Mar. 30, 2000; Ser. No. 60/197,324, filed 50
`Apr. 14,2000; Ser. No. 60/200,530, filed Apr. 27, 2000; Ser.
`No. 60/200,594, filed Apr. 28, 2000; and Ser. No. 60/202,
`087, filed May 4, 2000.
`This application also incorporates by reference the fol(cid:173)
`lowing publications: K. E. van Holde, Physical Biochemis- 55
`try (2n d ed. 1985); William Bains, Biotechnology from A to
`Z (1993); Richard P. Haugland, Handbook of Fluorescent
`Probes and Research Chemicals (6th ed. 1996); Joseph R.
`Lakowicz, Principles ofFluorescence Spectroscopy (2n d ed.
`1999); Bob Sinclair, Everything's Great When It Sits on a
`Chip: A Bright Future for DNA Arrays, 13 The Scientist,
`May 24, 1999, at 18; and Charles R. Cantor and Paul R.
`Schimmel, Biophysical Chemistry (1980).
`
`40
`
`60
`
`2
`for optical detection with improved read speed and/or
`signal-to-noise ratio. The apparatus and methods may be
`used with microplates, biochips, chromatography plates,
`microscope slides, and other substrates for high-throughput
`screening, genomics, SNPs analysis, pharmaceutical
`research and development, life sciences research, and other
`applications.
`
`BACKGROUND OF THE INVENTION
`
`Optical spectroscopy is the study of the interaction of
`light with matter. Typically, optical spectroscopy involves
`monitoring some property of light
`that is changed by its
`interaction with matter, and then using that change to
`characterize the components and properties of a molecular
`system. Recently, optical spectroscopy has been used in
`high-throughput screening procedures to identify candidate
`drug compounds.
`Optical spectroscopy is a broad term that describes a
`number of methods, such as absorption, luminescence (such
`as photoluminescence and chemiluminescence), scattering/
`reflectance, circular dichroism, optical rotation, and optical
`microscopy/imaging, among others. In turn, each of these
`terms describes a number of more closely related methods;
`for example photoluminescence includes fluorescence inten(cid:173)
`sity (FLINT), fluorescence polarization (FP), fluorescence
`resonance energy transfer (FRE1), fluorescence lifetime
`(FL1), total internal reflection fluorescence (TIRF), fluores(cid:173)
`cence correlation spectroscopy (FCS), fluorescence recov(cid:173)
`ery after photobleaching (FRAP), and their phosphorescence
`analogs, among others.
`Unfortunately, optical detection systems for use in optical
`spectroscopy suffer from a number of shortcomings. In
`particular, optical detection systems generally involve align(cid:173)
`ment of a sample and portions of an optical relay structure
`(such as an optics head) for directing light to and from the
`sample. Such alignment may be accomplished by physically
`moving the sample relative to the optical relay structure, or
`by physically moving the optical relay structure relative to
`the sample. Typically, such movement is followed by a
`waiting period before measurement to permit vibrations to
`subside. Time spent during alignment and subsequent wait(cid:173)
`ing periods is downtime because it is time during which data
`cannot be collected from the sample. Such downtime is
`especially significant in high-throughput screening, where
`tens or hundreds of thousands of samples must be aligned
`with an optical relay structure to conduct a particular study.
`In principle,
`the number of alignment steps can be
`reduced by reading simultaneously from a plurality of
`samples or from a larger area of a single sample. However,
`simultaneous reading typically will
`reduce intensities,
`because excitation light is distributed to a larger area and
`because the distance between the sample and optical relay
`structure is increased. Reduced intensities may decrease
`signal-to-noise ratios, decreasing reliability, especially with
`less intense nonlaser light sources.
`
`SUMMARY OF THE INVENTION
`
`The invention provides apparatus and methods for optical
`detection with improved read speed and/or signal-to-noise
`ratio.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIELD OF THE INVENTION
`The invention relates to optical detection. More
`particularly, the invention relates to apparatus and methods
`
`65
`
`FIG. 1 is a schematic view of a light detection device
`constructed in accordance with aspects of the invention,
`showing the device in use to read from a substrate.
`
`THERMO FISHER EX. 1032
`
`

`
`US 6,310,687 Bl
`
`3
`FIG. 2 is an alternative schematic VIew of the light
`detection device of FIG. 1.
`FIG. 3 is a schematic view of an alternative light detection
`device constructed in accordance with aspects of
`the
`invention, showing the device in use to read from a sub(cid:173)
`strate.
`FIG. 4 is an alternative schematic view of the light
`detection device of FIG. 3.
`FIGS. 5-7 are schematic views of other alternative light
`detection devices constructed in accordance with aspects of
`the invention.
`FIG. 8 is a partially exploded perspective view of yet
`another light detection device constructed in accordance
`with aspects of the invention, showing a transport module
`and an analysis module.
`FIG. 9 is a schematic view of an optical system from the
`analysis module of FIG. 8.
`FIG. 10 is a partially schematic perspective view of
`portions of the apparatus of FIG. 8.
`FIG. 11 is a schematic view of photoluminescence optical
`components from the optical system of FIG. 9.
`FIG. 12 is a schematic view of chemiluminescence optical
`components from the optical system of FIG. 9.
`
`DETAILED DESCRIPTION OF IRE
`INVENTION
`
`The invention provides apparatus and methods for optical
`detection with improved read speed and/or signal-to-noise
`ratio. These apparatus and methods may involve among
`others moving a sample substrate while simultaneously
`detecting light transmitted from one or more sample sites on
`the substrate by sequentially tracking the sample sites as
`they move. In this way, downtime associated with starting
`and stopping the sample substrate and with an inability to
`read during or immediately after moving the substrate may
`be reduced or eliminated. The following examples illustrate
`without limitation additional aspects of the invention.
`
`EXAMPLE 1
`FIG. 1 shows a light detection device 100 constricted in
`the invention. Device 100
`accordance with aspects of
`includes a stage 101, an examination region 102, and an
`optics head 104. Examination region 102 is delimited by a
`detection initiation position 106a and a detection termina(cid:173)
`tion position 106b. Stage 101 may be used to support a
`substrate 108 having a plurality of sample sites 110, such as
`a microplate and associated microplate wells, and optics
`head 104 may be used to direct light 112 to and/or from a 50
`sensed volume 114 positioned in a sample site located in the
`examination region. Specifically, light may be directed to the
`sample site from a light source 116, and/or light may be
`directed from the substrate to a detector 118. Typically, the
`examination region will be larger than the sensed volume,
`and the separation between adjacent/examined sample sites
`will be larger than the separation between the initiation
`position and the termination position. Suitable substrates,
`light sources, detectors, and optical relay structures for
`directing light to an optics head and substrate from a light
`source, and from a substrate and optics head to a detector are
`described below.
`Device 100 also includes a scanning mechanism 120
`configured to scan the substrate, so that device 100 may read
`from a plurality of positions on the substrate. In device 100,
`scanning mechanism 120 includes a reflective surface 122
`and is configured simultaneously to move (at least a portion
`
`5
`
`25
`
`4
`of) the optics head and substrate, preferably in a single
`direction. The optics head tracks the substrate between
`detection initiation position 106a and detection termination
`position 106b, and signal is collected continuously during an
`integration time over which there typically is no substantial
`relative motion between the optics head and the sample
`being analyzed. After the integration time, the position of the
`sensed volume (or optical beam) may be reset to the detec(cid:173)
`tion initiation position so that the sensed volume can track
`10 and detect from the next sample site on the substrate. If the
`reset
`time is small compared to the integration time,
`the
`percentage of time lost will be small. The scanning mecha(cid:173)
`nism improves read time by reducing the time that
`the
`detection optics spends over areas of the substrate that do not
`15 contain sample to be interrogated. (Any time spent over such
`areas can be considered downtime.) The scanning mecha(cid:173)
`nism also improves read time because the substrate moves
`continuously, more rapidly bringing new areas of the sub(cid:173)
`strate into position for reading, and because the need for a
`20 waiting period for vibrations to subside is reduced or elimi(cid:173)
`nated if the substrate does not jostle the samples by starting,
`stopping, or otherwise significantly changing speed. In this
`regard, the sample sites may move at a substantially constant
`speed, at least through the examination region.
`Device 100 may use any of various strategies to read from
`multiple sample sites. The device can read from the sample
`sites sequentially, one-by-one, as described above, or it can
`read from the sites in groups of two or more. Here, such
`reading groups may be parallel or perpendicular to the
`30 direction of reading, or a combination thereof. The device
`also can read from a first array in a first direction, move or
`offset in a second (typically perpendicular) direction, and
`then read again in the first direction from a second array
`parallel to the first array. Mechanisms for moving a sample
`35 substrate in one, two, or three directions are described in
`PCT Patent Application Ser. No. PCT/USOO/12277, filed
`May 3, 2000, which is incorporated herein by reference.
`Signal from samples on the (moving) substrate may be
`read by point-to-point reading or by constant velocity scan-
`40 ning. In point-to-point reading,
`the optics head is fixed
`relative to the substrate, as described above, while the signal
`from the detector is integrated for a desired period.
`In
`constant velocity scanning,
`the optical beam is moved
`relative to the substrate, while the signal from the detector
`45 is "binned" into pixels. The size of each pixel is simply the
`product of the scanning speed (relative to the substrate
`speed) and the integration time. For example, if the (relative)
`scanning speed is 10 mm per second and the integration time
`is 100 milliseconds, the pixel size is 1 mm.
`With this technique, a point-to-point reading detection
`system can read photoluminescent samples essentially as
`rapidly as a charge-coupled device (CCD)-based reading
`system with an equivalent light source and numerical aper(cid:173)
`ture. This is because the light source is the limitation, not the
`55 detector. A CCD is faster for chemiluminescence, because it
`collects the light emitted (which is not affected by detector
`area) from all samples simultaneously. The light output of
`each well
`is decreased in large-area photoluminescence,
`because the illumination per well
`is reduced, so that the
`60 increased speed resulting from collecting light from all wells
`in parallel is cancelled by the reduced illumination per well.
`nevertheless,
`the invention can be effective with
`fluorescence, phosphorescence, and chemiluminescence
`measurements, because for a given total read time, more
`65 time is spent
`integrating signal, and less time is spent
`aligning the optics with new samples. The invention is
`particularly effective with fluorescence polarization
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`US 6,310,687 Bl
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`5
`measurements, because good signal-to-noise ratios prefer(cid:173)
`ably involve collection of a minimum number of photons
`(e.g., 10,000) during the integration period, as described in
`U.S. patent application Ser. No. 09/349,733, which is incor(cid:173)
`porated herein by reference.
`FIG. 2 is an alternative view oflight detection device 100
`showing details of the optical relay structures. Here, light
`112 is directed from light source 116 (or equivalently from
`a fiber or other optics operatively connected to light source
`116) through a collimating (e.g., convex-plano) lens 124 and
`onto a beamsplitter 126, which directs a portion of the light
`toward substrate 108. Light emitted from the substrate is
`directed onto the beamsplitter, which transmits a portion of
`the light through a focusing (e.g., a plano-convex) lens 128
`toward detector 118 (or equivalently a fiber or other optics
`operatively connected to detector 118).
`Here, reflective element 122 (a parabolic section) may be
`moved to track the plate motion during integration, and then
`to "fly-back" quickly to the starting position for the next
`integration. If the input/output light 112 is collimated, the
`change in path length will not affect focus, spot size, or light
`collection, among others. The optics is reflective, which can
`improve efficiency, optical bandwidth, and cost relative to
`refractive optics. The moving element can be supported on
`nonfriction bearings, such as flexures (for example, on a
`four-bar linkage), because motion is small (-2 mm for a
`1536-well plate). Feedback can be provided to reduce posi(cid:173)
`tional error of the mirror. In fact, by measuring stage and
`mirror position and feeding back the error to the mirror
`drive, the stage and mirror can be locked together so that the
`mirror tracks the well location substantially exactly, even if
`the plate motion is not perfectly smooth. This has the
`significant advantage that substantially precise motion may
`be accomplished on a much lower mass object (the mirror,
`instead of the plate and its stage), so that bandwidth is higher
`and power requirements are lower.
`
`5
`
`6
`eter technique and a rotating polygon technique. The optics
`are substantially as described above for FIG. 2, except that
`a lens such as a plano-convex, converging, or other positive
`strength lens is used between the scanning mechanism and
`the substrate for field flattening.
`The primary drawing in FIG. 4 illustrates a galvanometer
`technique. Here, driven by a galvanometer-type movement,
`a mirror 220 pivots through a small angle and then returns
`to its start position to repeat
`the cycle. Suitable drivers
`10 include galvanometers, voice-coil drivers, and piezo drivers.
`The mirror and driver typically are supported by nonfriction
`bearings, which may include springs, torsion springs, and/or
`flexures. A lack of stick-slip enables precise, low-power
`positioning. The system can be resonant, meaning that the
`15 compliance of the bearings resonates with the combined
`mass of the mirror and driver. If the system is resonant,
`power requirements will drop significantly. Feedback can be
`provided as above to reduce positional error of the mirror.
`The inset in FIG. 4 illustrates a rotating polygon tech-
`20 nique. Here, instead of scanning a mirror back-and-forth as
`above, a polygonal mirror 230 (or section 232 thereof)
`rotates in synchrony with the stage. The motor drive may be
`much easier: if the mirrors are curved, or if an optic is added,
`the motion may be at constant angular velocity. To reduce
`25 dead time between integrations, the polygon should be large
`compared to the collimated beam. (Dead time occurs when
`the beam is on two facets of the mirror at once.)
`With both the galvanometer and rotating polygon
`30 techniques, the focused spot tends to follow an arc. If the
`plate is planar, resulting difficulties may be corrected by
`effectively increasing the radius of curvature of the arc by
`adding a field-flattening optic, by offsetting the axis of
`rotation of the galvanometer, and/or by providing a rotating
`35 polygon with curved faces. Whether corrected or not, the arc
`will
`track the sample site in the same direction over the
`distance scale of the examination region.
`
`EXAMPLE 2
`FIG. 3 shows an alternative light detection device 200
`constructed in accordance with aspects of the invention. 40
`Device 200 includes a stage 201, an examination site 202
`delimited as above, and an optics head 204 for directing light
`206 to and/or from a substrate 208 positioned in the exami(cid:173)
`nation site. Device 200 also includes a scanning mechanism
`210 configured to scan the substrate. In device 200,
`the 45
`scanning mechanism is configured to move the substrate
`while holding the optics head fixed. More specifically, the
`scanning mechanism is configured to rotate rather
`than
`translate. Scanning mechanism 212 may include a galva(cid:173)
`nometer mirror and/or a rotating polygon mirror for match-
`ing illumination and/or detection with particular areas of the
`substrate. Galvanometer mirrors include small planar or
`convex mirrors attached to the rotating coil of a galvanom(cid:173)
`eter to move a spot of reflected light, among others. Rotating
`polygon mirrors include a polygonal mirror attached to a 55
`driver to move a spot of reflected light, among others.
`Device 200 may be used with any light source, although
`nonlaser light sources, such as arc lamps or LEDs, present
`special difficulties. This is because the distance between the
`source and detector may be relatively long, which may result
`in lower efficiencies with nonlaser light sources. Some of the
`difficulty may be overcome by using a high color tempera(cid:173)
`ture continuous light source, as described in U.S. patent
`application Ser. No. 09/349,733, which is incorporated
`herein by reference.
`FIG. 4 shows an alternative view of light detection device
`200, illustrating several techniques, including a galvanom-
`
`EXAMPLE 3
`FIGS. 5-6 show other alternative light detection devices
`constructed in accordance with aspects of the invention.
`These devices involve scanning an aperture over a larger
`area detector/source. In these (and other) embodiments, the
`light may not be collimated as it goes through the scanning
`mechanism.
`FIG. 5 shows a first pair of embodiments involving
`scanning an aperture. If the detector can accommodate the
`entire motion of the scanned location (e.g., an area of 2.25
`mmx4.5 mm for a 1536-well microplate), which is true with
`a photomultiplier tube (PMT), and if the source can illumi(cid:173)
`nate it,
`then only an aperture need be scanned. This is
`accomplished by imaging a small area of the plate adjacent
`the well being measured onto a second "aperture plate." The
`aperture plate is moved in synchrony with the sample plate,
`but in the opposite direction, so that light to and from only
`one well can make it
`through the aperture.
`If the lens
`demagnifies by a factor 11m, then the aperture plate should
`move at a speed m times the sample plate speed. The
`subsequent optics has much-relaxed imaging requirements
`because there is little or no possibility of cross-talk. The
`aperture plate also could have more than one set of associ-
`ated optics to increase throughput (requiring multiple imag(cid:173)
`ing elements) or to provide "quick-change" capability for
`different wavelengths, excitations, etc. The dichroic mirror
`65 can pivot to reduce the illuminated area requirement.
`A sample plate or other substrate can be imaged onto an
`"aperture plate" refractively or reflectively, among others. A
`
`50
`
`60
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`7
`plate can be imaged refractively using a lens. A plate can be
`imaged reflectively (with advantages as mentioned above)
`using a mirror, such as a section of an ellipse. The mirror
`may be dichroic, which can eliminate all lenses and greatly
`increase bandwidth; this permits the focus to be adjusted
`without moving the aperture plate or optics Gust the imaging
`unit), so that
`the light source(s) and detector(s) can be
`mounted at the optics head, eliminating the cost and light
`loss associated with fiber optics. Again, mirrors can be
`scanned or pivoted to reduce illumination requirements.
`FIG. 6 shows a second pair of embodiments involving
`scanning an aperture. The imaging optics (mirror or lens)
`can be rotated, or a prism inside the imaging optics can be
`rotated. Alternatively, the techniques described above can be
`used with an ellipsoidal mirror, with or without demagnifi(cid:173)
`cation.
`
`EXAMPLE 4
`
`FIG. 7 shows yet another alternative device constructed in
`accordance with aspects of the invention, using a Digital
`Mirror Device (DMD). This device has a large array of very
`small (10-20 micron), very fast mirrors that can be rotated
`under electronic control. Placed in an image plane, they can
`be used to control the area that is reflected into the optics. A
`suitable DMD (used for video projectors) may be obtained
`commercially from Texas Instruments Inc. (Dallas, Tex.).
`
`EXAMPLE 5
`
`The apparatus and methods for optical detection provided
`by the invention can be used in a large variety of optical
`systems and for a large variety of optical applications. This
`example describes a preferred system, namely a multi-mode
`high-throughput
`light-detection system for analyzing
`samples.
`FIG. 8 shows such a system 350, which includes a
`transport module 352 and an analysis module 354 capable of
`detecting and analyzing light. The transport module includes
`I/O sites 356, a transfer site 358, and mechanisms (not
`visible) for transporting sample holders between the I/O and
`transfer sites, as described above. The analysis module
`includes a housing 360, a moveable control unit 362, an
`optical system (not visible), and a transport mechanism 364.
`The housing may be used to enclose the analysis module,
`protecting both the user and components of the module, and
`may be used as a fixed reference point to describe the
`motions of any moveable portions of the apparatus, such as
`a scanning optics head. The control unit may be used to
`operate the module manually and/or
`robotically, as
`described in U.S. Pat. No. 6,025,985, which is incorporated
`herein by reference. The optical system and transport
`mechanisms are described in subsequent sections.
`FIGS. 9-12 show an optical system (and related
`components) 390 for use in system 350. The optical system
`may include components for generating and/or detecting
`light, and for transmitting light to and/or from a sample.
`These components may include (1) a stage for supporting
`the sample, (2) one or more light sources for delivering light
`to the sample, (3) one or more detectors for receiving light
`transmitted from the sample and converting it to a signal, (4)
`first and second optical relay structures for relaying light
`between the light source, sample, and detector, and/or (5) a
`processor for analyzing the signal from the detector. System
`components may be chosen to optimize speed, sensitivity,
`and/or dynamic range for one or more assays. For example,
`optical components with low intrinsic luminescence may be
`used to enhance sensitivity in luminescence assays by reduc-
`
`5
`
`10
`
`8
`ing background. System components also may be shared by
`different assays, or dedicated to particular assays. For
`example, steady-state photoluminescence assays may use a
`continuous light source, time-resolved photoluminescence
`assays may use a time-varying light source, and chemilu(cid:173)
`minescence assays may not use a light source. Similarly,
`steady-state and time-resolved photoluminescence assays
`may both use a first detector, and chemiluminescence assays
`may use a second detector.
`Optical system 390 includes (a) a photoluminescence
`optical system, and (b) a chemiluminescence optical system,
`as described below. Further aspects of the optical system are
`described in the following patent applications, which are
`incorporated herein by reference: U.S. patent application
`15 Ser. No. 09/160,533, filed Sep. 24, 1998; U.S. patent appli(cid:173)
`cation Ser. No. 09/349,733, filed Jul. 8, 1999; PCT Patent
`Application Ser. No. PCT/US99/16287, filed Jul. 26, 1999;
`and PCT Patent Application Ser. No. PCT/USOO/04543,
`filed Feb. 22, 2000.
`20 a. Photoluminescence Optical System
`FIGS. 9-11 show the photoluminescence (or
`incident
`light-based) optical