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`US007148043B2
`
`c12) United States Patent
`Kordunsky et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 7,148,043 B2
`Dec. 12, 2006
`
`(54) SYSTEMS AND METHODS FOR
`FLUORESCENCE DETECTION WITH A
`MOVABLE DETECTION MODULE
`
`(75)
`
`Inventors: Igor Kordunsky, Newton, MA (US);
`Jeffrey A. Goldman, Acton, MA (US);
`Michael J. Finney, San Francisco, CA
`(US)
`
`(73) Assignee: Bio-Rad Laboratories, Inc., Hercules,
`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.: 10/431,708
`
`(22) Filed:
`
`May 8, 2003
`
`(65)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`Prior Publication Data
`
`US 2004/0224317 Al
`
`Nov. 11, 2004
`
`Int. Cl.
`C12P 19134
`(2006.01)
`C12Q 1168
`(2006.01)
`U.S. Cl. ......................... 435/91.2; 435/6; 435/91.1
`Field of Classification Search ............. 250/458.1;
`435/6, 91.1, 91.2; 422/82.05, 82.07
`See application file for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`2/1993 Hearst et al.
`5,184,020 A
`5/1993 Gelfand et al.
`5,210,015 A
`5/1995 Zaun et al.
`5,415,839 A
`12/1995 Blumenfeld et al.
`5,473,437 A
`5,585,242 A * 12/1996 Bouma et al.
`................. 435/6
`5,736,333 A
`4/1998 Livak et al.
`5,928,907 A
`7/1999 Woudenberg et al.
`5,972,716 A
`10/1999 Ragusa et al.
`5,994,056 A
`11/1999 Higuchi
`
`6,015,674 A
`6,024,920 A
`6,043,880 A
`6,140,054 A
`6,144,448 A
`6,174,670 Bl
`6,197,575 Bl
`6,337,435 Bl
`6,359,284 Bl *
`6,369,893 Bl
`6,569,631 Bl*
`6,818,437 Bl
`2002/0064780 Al*
`2003/0015668 Al*
`
`1/2000 Woudenberg et al.
`212000 Cunanan
`3/2000 Andrews et al.
`10/2000 Wittwer et al.
`11/2000 Mitoma
`1/2001 Wittwer et al.
`3/2001 Griffith et al.
`1/2002 Chu et al.
`3/2002 Hayashi et al. .......... 250/458.l
`412002 Christel et al.
`5/2003 Pantoliano et al.
`11/2004 Garnbini et al.
`512002 Gold et al.
`. ................... 435/6
`1/2003 Montagu ................. 250/458.l
`
`.......... 435/7.1
`
`OTHER PUBLICATIONS
`
`PCT International Preliminary Report on Patentability for PCT/
`US04/14566.
`* cited by examiner
`Primary Examiner-Young J. Kim
`(74) Attorney, Agent, or Firm-Townsend and Townsend
`and Crew LLP
`
`(57)
`
`ABSTRACT
`
`A fluorescence detection apparatus for analyzing samples
`located in a plurality of wells in a thermal cycler and
`methods of use are provided. In one embodiment, the
`apparatus includes a support structure attachable to the
`thermal cycler and a detection module movably mountable
`on the support structure. The detection module includes one
`or more channels, each having an excitation light generator
`and an emission light detector both disposed within the
`detection module. When the support structure is attached to
`the thermal cycler and the detection module is mounted on
`the support structure, the detection module is movable so as
`to be positioned in optical communication with different
`ones of the plurality of wells. The detection module is
`removable from the support structure to allow easy replace(cid:173)
`ment.
`
`18 Claims, 7 Drawing Sheets
`
`THERMO FISHER EX. 1026
`
`

`
`U.S. Patent
`
`Dec. 12, 2006
`
`Sheet 1 of 7
`
`US 7,148,043 B2
`
`122
`
`112
`
`121
`
`FIG. I
`
`THERMO FISHER EX. 1026
`
`

`
`nee. 12, 2006
`
`Sbeet 2 of '1
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`000000 00 0000
`000000000000
`0000 0000000 ~
`
`FIG. 2
`
`THERMO FISHER EX. 1026
`
`

`
`U.S. Patent
`
`Dec. 12, 2006
`
`Sheet 3 of 7
`
`US 7 ,148,043 B2
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`THERMO FISHER EX. 1026
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`

`
`U.S. Patent
`
`Dec. 12, 2006
`
`Sheet 4 of 7
`
`US 7,148,043 B2
`
`420
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`420
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`234
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`
`THERMO FISHER EX. 1026
`
`

`
`U.S. Patent
`
`Dec. 12, 2006
`
`Sheets of 7
`
`US 7,148,043 B2
`
`606
`
`600
`
`634
`
`628
`
`636
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`602
`
`FIG. 6
`
`THERMO FISHER EX. 1026
`
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`
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`
`702
`
`THERMO FISHER EX. 1026
`
`

`
`U.S. Patent
`
`Dec. 12, 2006
`
`Sheet 7 of 7
`
`US 7,148,043 B2
`
`PREPARE REACTION VESSELS
`WITH SAMPLES TO BE ANA'L YZED
`
`+
`
`MOUNT DETECTION MODULE
`ON SHUTILE
`
`PLACE REACTION VESSELS IN
`SAMPLE WELLS
`
`CLOSE LID AND PLACE UNIT
`IN THERMAL CYCLER BASE
`
`+
`•
`+
`•
`
`;-800
`802
`
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`
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`808
`
`r
`
`810
`
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`
`812
`
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`
`814
`
`CALIBRATE DETECTION MODULE
`
`PERFORM PCR CYCLE
`__________ j __________
`SCAN AND INTERROGATE REACTION VESSELS
`v-816a
`
`POSITION DETECTION MODULE IN
`OPTICAL COMMUNICATION WITH WELLS
`
`ACTIVATE LED
`
`+
`•
`
`DETECT FLUORESCENT RESPONSE
`
`v- 816b
`
`816c
`
`r
`
`L----------------------------
`FIG. 8
`
`THERMO FISHER EX. 1026
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`

`
`US 7,148,043 B2
`
`1
`SYSTEMS AND METHODS FOR
`FLUORESCENCE DETECTION WITH A
`MOVABLE DETECTION MODULE
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates in general to fluorescence
`detection systems and in particular to a fluorescence detec(cid:173)
`tion system having a movable excitation/detection module
`for use with a thermal cycler.
`Thermal cyders are known in the art. Such devices are
`used in a variety of processes for creation and detection of
`various molecules of interest, e.g., nucleic acid sequences, in
`research, medical, and industrial fields. Processes that can be
`performed with conventional thermal cyders include but are
`not limited to amplification of nucleic acids using proce(cid:173)
`dures such as the polymerase chain reaction (PCR). Such
`amplification processes are used to increase the amount of a
`target sequence present in a nucleic acid sample.
`Numerous techniques for detecting the presence and/or
`concentration of a target molecule in a sample processed by
`a thermal cycler are also known. For instance, fluorescent
`labeling may be used. A fluorescent label (or fluorescent
`probe) is generally a substance which, when stimulated by
`an appropriate electromagnetic signal or radiation, absorbs
`the radiation and emits a signal (usually radiation that is
`distinguishable, e.g., by wavelength, from the stimulating
`radiation) that persists while the stimulating radiation is
`continued, i.e. it fluoresces. Some types of fluorescent
`probes are generally designed to be active only in the
`presence of a target molecule (e.g., a specific nucleic acid
`sequence), so that a fluorescent response from a sample
`signifies the presence of the target molecule. Other types of
`fluorescent probes increase their fluorescence in proportion
`to the quantity of double-stranded DNA present in the
`reaction. These types of probes are typically used where the
`amplification reaction is designed to operate only on the
`target molecule.
`Fluorometry involves exposing a sample containing the
`fluorescent label or probe to stimulating (also called exci(cid:173)
`tation) radiation, such as a light source of appropriate
`wavelength, thereby exciting the probe and causing fluores(cid:173)
`cence. The emitted radiation is detected using an appropriate
`detector, such as a photodiode, photomultiplier, charge(cid:173)
`coupled device (CCD), or the like.
`Fluorometers for use with fluorescent-labeled samples are
`known in the art. One type of fluorometer is an optical
`reader, such as described by Andrews et al. in U.S. Pat. No.
`6,043,880. A sample plate containing an array of samples is
`inserted in the optical reader, which exposes the samples to 50
`excitation light and detects the emitted radiation. The use(cid:173)
`fulness of optical readers is limited by the need to remove
`the sample plate from the thermal cycler, making it difficult
`to monitor the progress of amplification.
`One improvement integrates the optical reader with a 55
`thermal cycler, so that the sample plate may be analyzed
`without removing it from the thermal cycler or interrupting
`the PCR process. Examples of such combination devices are
`described in U.S. Pat. Nos. 5,928,907, 6,015,674, 6,043,880,
`6,144,448, 6,337,435, and 6,369,863. Such combination 60
`devices are useful in various applications, as described, e.g.,
`in U.S. Pat. Nos. 5,210,015, 5,994,056, 6,140,054, and
`6,174,670.
`Existing fluorometers suffer from various drawbacks. For
`instance, in some existing designs, different light sources
`and detectors are provided for different sample wells in the
`array. Variations among the light sources and/or detectors
`
`2
`lead to variations in the detected fluorescent response from
`one well to the next. Alternatively, the light source and/or
`detector may be arranged in optical communication with
`more than one of the wells, with different optical paths to
`and/or from each well. Due to the different optical paths, the
`detected fluorescent response varies from one sample well to
`the next. To compensate for such variations, the response for
`each sample well must be individually calibrated. As the
`number of sample wells in an array increases, this becomes
`10 an increasingly time-consuming task, and errors in calibra(cid:173)
`tion may introduce significant errors in subsequent measure(cid:173)
`ments.
`In addition, existing fluorometers generally are designed
`such that the light sources and detectors are fixed parts of the
`15 instrument. This limits an experimenter's ability to adapt a
`fluorometer to a different application. For instance, detecting
`a different fluorescent label generally requires using a dif(cid:173)
`ferent light source and/or detector. Many existing fluorom(cid:173)
`eters make it difficult for an experimenter to reconfigure
`20 light sources or detectors, thus limiting the variety of fluo(cid:173)
`rescent labels that may be used.
`It is also difficult to perform concurrent measurements of
`a number of different fluorescent labels that may be present
`in a sample (or in different samples). As described above, to
`25 maximize the data obtained in an assay, experimenters often
`include multiple fluorescent labeling agents that have dif(cid:173)
`ferent excitation and/or emission wavelengths. Each label(cid:173)
`ing agent is adapted to bind to a different target sequence, in
`principle allowing multiple target sequences to be detected
`30 in the same sample. Existing fluorometers, however, do not
`facilitate such multiple-label experiments. Many fluorom(cid:173)
`eters are designed for a single combination of excitation and
`emission wavelengths. Others provide multiple light sources
`and detectors to allow detection of multiple labels; however,
`35 these configurations often allow only one label to be probed
`at a time because the excitation wavelength of one label may
`overlap the emission wavelength of another label; excitation
`light entering the detector would lead to incorrect results.
`Probing multiple labels generally cannot be done in parallel,
`40 slowing the data collection process.
`Therefore, an improved fluorometer for a thermal cycler
`that overcomes these disadvantages would be desirable.
`
`BRIEF SUMMARY OF THE INVENTION
`
`45
`
`Embodiments of the present invention provide fluores(cid:173)
`cence detection in a thermal cycling apparatus. According to
`one aspect of the invention, a fluorescence detection appa(cid:173)
`ratus for analyzing samples located in a plurality of wells in
`a thermal cycler includes a support structure attachable to
`the thermal cycler and a detection module movably mount-
`able on the support structure. The detection module includes
`an excitation light generator and an emission light detector,
`both disposed within the detection module. When the sup(cid:173)
`port structure is attached to the thermal cycler and the
`detection module is mounted on the support structure, the
`detection module is movable so as to be positioned in optical
`communication with different ones of the plurality of wells.
`According to another aspect of the invention, the detec(cid:173)
`tion module may include two or more excitation light
`generators and two or more emission light detectors
`arranged to form two or more excitation/detection pairs. In
`one embodiment, the excitation/detection pairs are arranged
`such that each excitation/detection pair is simultaneously
`65 positionable in optical contact with a different one of the
`plurality of wells. In an alternative embodiment, excitation/
`detection pairs are arranged such that when a first one of the
`
`THERMO FISHER EX. 1026
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`

`
`US 7,148,043 B2
`
`3
`excitation/detection pairs is positioned in optical contact
`with any one of the plurality of wells, a different one of the
`excitation/detection pairs is not in optical contact with any
`one of the plurality of wells. In some embodiments, the
`detection module is detachably mounted on the support
`structure, thereby enabling a user to replace the detection
`module with a different detection module.
`According to yet another aspect of the invention, a
`method for detecting the presence of a target molecule in a
`solution is provided. A plurality of samples is prepared, each
`sample containing a fluorescent probe adapted to bind to a
`target molecule. Each sample is placed in a respective one of
`a number of sample wells of a thermal cycler instrument, the
`thermal cycler instrument having a detection module mov(cid:173)
`ably mounted therein, the detection module including an
`excitation/detection channel, the excitation/detection chan(cid:173)
`nel including an excitation light generator disposed within
`the detection module and an emission light detector disposed
`within the detection module. The thermal cycler instrument
`is used to stimulate a reaction, and the sample wells are
`scanned to detect a fluorescent response by moving the
`detection module and activating the excitation/detection
`channel. During the scanning, the detection module is
`moved such that the excitation/detection channel is sequen(cid:173)
`tially positioned in optical communication with each of the 25
`plurality of sample wells. Where the detection module
`includes multiple excitation/detection pairs or channels,
`channels may be active in parallel or sequentially.
`The following detailed description together with the
`accompanying drawings will provide a better understanding 30
`of the nature and advantages of the present invention.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a perspective view of a thermal cycling appa(cid:173)
`ratus according to an embodiment of the present invention;
`FIG. 2 is an exploded view of a lid assembly for a thermal
`cycling apparatus according to an embodiment of the present
`invention;
`FIG. 3 is a bottom view of a fluorometer assembly for a
`thermal cycling apparatus according to an embodiment of
`the present invention;
`FIG. 4 is a top view of detection module according to an
`embodiment of the present invention;
`FIGS. SA-B are bottom views of detection modules
`according to alternative embodiments of the present inven(cid:173)
`tion;
`FIG. 6 is a schematic diagram of an excitation/detection
`pair for a detection module according to an embodiment of
`the present invention;
`FIG. 7 is a block diagram illustrating electrical connec(cid:173)
`tions for a lid assembly for a thermal cycling apparatus
`according to an embodiment of the present invention; and
`FIG. 8 is a flow diagram of a process for using a thermal
`cycler having a fluorescence detection system according to
`an embodiment of the present invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`An exemplary apparatus embodiment of the present
`invention will be described with reference to the accompa(cid:173)
`nying drawings, in which like reference numerals indicate
`corresponding parts. Methods of using the apparatus will
`also be described. It is to be understood that embodiments
`shown and described herein are illustrative and not limiting
`of the invention.
`
`4
`
`I. Exemplary Apparatus
`FIG. 1 is a perspective view of a thermal cycling appa(cid:173)
`ratus 100 according to an embodiment of the present inven(cid:173)
`tion. Apparatus 100 consists of a base unit 110 and a lid
`assembly 112. Base unit 110, which may be of conventional
`design, provides power and control functions for a thermal
`cycling process via conventional electronic components (not
`shown), such as programmable processors, clocks, and the
`like. Base unit 110 also provides a user interface 116 that
`10 may include a keypad 118 and an LCD display screen 120,
`enabling a user to control and monitor operation of the
`thermal cycler. Base unit 110 connects to an external power
`source (e.g., standard 120 V ac power) via a power cable
`121. Some examples of base unit 110 include the DNA
`15 Engine®, Dyad™, and Tetrad™ thermal cyders sold by MJ
`Research, Inc., assignee of the present application.
`Lid assembly 112 includes a sample unit and a fluores(cid:173)
`cence detection apparatus, disposed within a lid 122; these
`components will be described below. Lid 122 has a handle
`20 124 to aid in its placement on and removal from base unit
`110, and ventilation holes 126. Lid 122 provides optical and
`thermal isolation for the components inside lid assembly
`112.
`FIG. 2 is an exploded view of the inside of lid assembly
`112. Shown are a sample unit 202, a lid heater 204, and a
`fluorometer assembly 206. Sample unit 202 contains a
`number of sample wells 210 arranged in a regular array (e.g.,
`an 8x12 grid). In one embodiment, each sample well 210
`holds a removable reaction vessel (not shown), such as a
`tube, that contains a nucleic acid sample to be tested,
`together with appropriate PCR reactants (buffers, primers
`and probes, nucleotides, and the like) including at least one
`fluorescent label or probe adapted to bind to or otherwise
`respond to the presence of a target nucleic acid sequence.
`35 The reaction vessels are advantageously provided with
`transparent sample caps (not shown) that fit securely over
`the tops of the vessels to prevent cross-contamination of
`samples or spillage during handling. Reaction vessels may
`also be sealed in other ways, including the use of films such
`40 as Microseal®B (made by MJ Research, Inc.), wax products
`such as Chill-out™ (made by MJ Research, Inc.), or mineral
`oil. In an alternative configuration, a removable sample tray
`(not shown) that holds one or more distinct samples at
`locations corresponding to sample wells 210 is used. The
`45 sample tray may also be sealed in any of the ways described
`above.
`Sample unit 202 also includes heating elements (e.g.,
`Peltier-effect thermoelectric devices), heat exchange ele(cid:173)
`ments, electrical connection elements for connecting the
`50 heating elements to base unit 110, and mechanical connec(cid:173)
`tion elements. These components (not shown) may be of
`conventional design. Sample unit 202 also provides electri(cid:173)
`cal connections for lid heater 204 and fluorometer assembly
`206 via multiwire cables 212, which are detachably con-
`55 nected to connectors 214.
`Lid heater 204 has holes 220 therethrough, matching the
`size and spacing of the sample wells 210, and electronically
`controlled heating elements (not shown). Lid heater 204 is
`coupled to lid 122. The coupling mechanism (not shown) is
`60 advantageously movable (e.g., lid heater 204 may be
`attached to lid 122 by a hinge) in order to provide access to
`fluorometer assembly 206 when lid 122 is removed from
`sample unit 202. When lid 122 is in place on sample unit
`202, supports 224 hold lid heater 204 in position. Lower
`65 portions 226 of supports 224 are advantageously designed to
`compress lid heater 204 toward sample unit 202, thereby
`reducing the possibility of sample evaporation during opera-
`
`THERMO FISHER EX. 1026
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`
`US 7,148,043 B2
`
`5
`tion of apparatus 100. This compression also allows reaction
`vessels of different sizes to be used. Lid heater 204 is used
`to control the temperature of the sample caps (or other
`sealants) of reaction vessels sample wells 210, in order to
`prevent condensation from forming on the caps during
`thermal cycling operation.
`Lid heater 204 advantageously includes one or more
`calibration elements 222 positioned between selected ones
`of holes 220 or in other locations away from the holes, such
`as near the periphery of lid heater 204. Calibration elements
`222 provide a known fluorescence response and may be used
`to calibrate fluorescence detectors in fluorometer assembly
`206. Calibration elements 222 may be made, e.g., of a
`fluorescent coating on a glass or plastic substrate, or they
`may consist of a plastic with a dye impregnated in it,
`fluorescent glass, or a fluorescent plastic such as polyether(cid:173)
`imide (PEI). Neutral-density or other types of filters may be
`placed over the fluorescent material in order to avoid satu(cid:173)
`rating the fluorescence detectors. In general, any material
`may be used, provided that its fluorescence characteristics
`are sufficiently stable over time with the application of light
`(photo-bleaching) and heat. To the extent practical, the effect
`of temperature on the fluorescence response is advanta(cid:173)
`geously minimized. Where multiple calibration elements
`222 are provided, different materials may be used for 25
`different ones of the calibration elements. In an alternative
`embodiment, lid heater 204 may be omitted, and calibration
`elements 222 may be disposed on the surface of sample unit
`202.
`Sample unit 202 and lid heater 204 may be of conven(cid:173)
`tional design. Examples of suitable designs include sample
`unit and lid heater components of the various Alpha™
`modules sold by MJ Research, Inc., assignee of the present
`application.
`Fluorometer assembly 206 includes a support frame or 35
`platform 230 fixedly mounted inside lid 122. Movably
`mounted on the underside of support frame 230 is a shuttle
`232, which holds a detection module 234. Shuttle 232 is
`movable in two dimensions so as to position detection
`module 234 in optical communication with different ones of 40
`the sample wells 210 in sample unit 202 through the
`corresponding holes 220 in lid heater 204. Support frame
`230 and supports 224 are advantageously dimensioned such
`that when lid 122 is positioned in base unit 110 and closed,
`detection module 234 is held in close proximity to lid heater 45
`204; one of skill in the art will appreciate that this arrange(cid:173)
`ment reduces light loss between the sample wells and the
`detection module.
`FIG. 3 is a bottom view of fluorometer assembly 206,
`showing a movable mounting of shuttle 232 and detection
`module 234. In this embodiment, translation stages driven
`by stepper motors are used to move the shuttle 232, to which
`detection module 234 is detachably coupled, to a desired
`position. Specifically, support platform 230 has an x-axis
`stepper motor 302 and a lead screw 304 attached thereto.
`Stepper motor 302 operates to turn lead screw 304, thereby
`moving a translation stage 306 along the x direction (indi(cid:173)
`cated by arrow). Limit switches 308 are advantageously
`provided to restrict the motion of translation stage 306 to an
`appropriate range, large enough to allow detection module 60
`234 to be placed in optical contact with any of the wells
`while preventing translation stage 306 from contacting other
`system components, such as stepper motor 302.
`Translation stage 306 has a y-axis stepper motor 316 and
`a lead screw 318 mounted thereon. Stepper motor 316 65
`operates to turn lead screw 318, thereby moving shuttle 232
`along the y direction (indicated by arrow). Limit switches
`
`6
`320 are advantageously provided to restrict the motion of
`shuttle 232 to an appropriate range, large enough to allow
`detection module 234 to be placed in optical contact with
`any of the wells, while preventing shuttle 232 from contact(cid:173)
`ing other system components, such as stepper motor 316.
`Stepper motors 302, 316, lead screws 304, 318, and limit
`switches 308, 320 may be of generally conventional design.
`It will be appreciated that other movable mountings may be
`substituted. For example, instead of directly coupling the
`10 motors to the lead screws, indirect couplings such as chain
`drives or belt drives may be used. Chain drives, belt drives,
`or other drive mechanisms may also be used to position the
`detection module without lead screws, e.g., by attaching a
`translation stage to the chain, belt, or other drive mechanism.
`15 Other types of motors, such as servo motors or linear motors,
`may also be used. Different drive mechanisms may be used
`for different degrees of freedom.
`Shuttle 232 holds detection module 234 via connectors
`330, 331. Connectors 330, 331 which may vary in design,
`20 are configured to support and align detection module 234 on
`the underside of shuttle 232. The connectors are advanta-
`geously adapted to allow easy insertion and removal of
`detection module 234, to facilitate replacement of the detec(cid:173)
`tion module. In one embodiment, connectors 330 provide
`mounting for a cylindrical member (not shown) that pivot(cid:173)
`ably holds an edge of detection module 234, while connec-
`tors 331 include ball plungers mounted on shuttle 232 that
`are insertable into corresponding receptacles on detection
`module 234. Electrical connections (not shown) between
`30 shuttle 232 and detection module 234 may also be provided,
`as will be described below.
`FIG. 4 is a top view of detection module 234. Detection
`module 234 includes fittings 420 that couple to correspond(cid:173)
`ing connectors 330 on the underside of shuttle 232, thereby
`securing detection module 234 in place so that it moves as
`a unit with shuttle 232. Detection module 234 also includes
`an electrical connector 424 that couples to a corresponding
`electrical connector on the underside of shuttle 232, thereby
`allowing control and readout signals to be provided to and
`obtained from detection module 234.
`FIG. SA is a bottom view of one embodiment of detection
`module 234, showing four openings S02, S04, S06, S08 for
`four independently controlled fluorescent excitation/detec(cid:173)
`tion channels (also referred to as "excitation/detection
`pairs") arranged inside the body of detection module 234.
`Examples of excitation/detection channels will be described
`below. The spacing of openings S02, S04, S06, S08 corre(cid:173)
`sponds to the spacing of sample wells 210. Thus, when
`opening S02 is placed in optical communication with one of
`50 the sample wells 210, openings S04, S06, and S08 are each
`in optical communication with a different one of the sample
`wells 210. Openings S02, S04, S06, S08 may simply be holes
`through the bottom surface of detection module 234, or they
`may be made of any substance that has a high degree of
`55 transparency to the excitation and detection light wave(cid:173)
`lengths of their respective channels.
`FIG. SB is a bottom view of a detection module 234'
`according to an alternative embodiment of the invention. In
`this embodiment, four openings S12, S14, S16, S18 are
`provided, but they are arranged in a staggered fashion so that
`only one opening at a time may be in optical communication
`with any of the sample wells. This configuration is useful for
`reducing cross-talk between the excitation/detection pairs.
`FIG. 6 is a schematic diagram illustrating a configuration
`of optical elements for an excitation/detection channel (or
`excitation/detection pair) 600 according to an embodiment
`of the invention. Detection module 234 may include one or
`
`THERMO FISHER EX. 1026
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`

`
`US 7,148,043 B2
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`7
`8
`more instances of excitation/detection pair 600, each of
`LEDs provide a compact and reliable light source, use of
`other types of coherent or incoherent light sources, such as
`which provides an independent fluorescence detection chan(cid:173)
`nel. Excitation/detection pair 600 is arranged inside opaque
`laser diodes, flash lamps, and so on, is not precluded.
`walls 602, which provide optical isolation from other exci(cid:173)
`Similarly, the detectors are not limited to photodiodes; any
`type of photodetector may be substituted, including photo(cid:173)
`tation/detection pairs that may be included in detection
`module 234, as well as from external light sources. An
`multipliers and charge-coupled devices (CCDs). Each exci(cid:173)
`excitation light path 604 includes a light-emitting diode
`tation/detection pair is advantageously configured as a self(cid:173)
`(LED) or other light source 606, a filter 608, a lens 610, and
`contained assembly, requiring only external electrical
`a beam splitter 612. A detection light path 620 includes beam
`connections to make it operational. Because the length of the
`splitter 612, a filter 624, a lens 626, and a photodiode or
`10 excitation and detection optical paths do not vary from one
`other photodetector 628. Beam splitter 612 is advanta(cid:173)
`experiment to the next, it is desirable to fixedly mount and
`optimize the various optical components of each excitation/
`geously selected to be highly transparent to light of the
`detection pair 600 inside detection module 234 during
`excitation wavelength and highly reflective of light at the
`manufacture so that further adjustments during operation are
`detection (fluorescent response) wavelength.
`The components of excitation light path 604 are arranged 15 not required.
`to direct excitation light of a desired wavelength into a
`FIG. 7 is a block diagram illustrating electrical connec(cid:173)
`reaction vessel 616 held in a sample well 210 of sample
`tions for lid assembly 112. A main processing board 702 is
`block 202. The desired wavelength depends on the particular
`mounted in lid assembly 112. Main processing board 702
`fluorescent labeling agents included in reaction vessel 616
`includes a primary signal processor 704, a stepper motor
`and is controlled by selection of an appropriate LED 606 and
`20 driver unit 706, a connection 708 for electrical power, and a
`filter 608. Optical communication between the excitation/
`connection 710 for an external computer (e.g., a personal
`detection pair 600 and reaction vessel 616 is provided by
`computer, or PC). Main processing board 702 also provides
`opening 502 in opaque walls 602 and a hole 220 through lid
`connectors 214 for cables 212 that provide transmission of
`heater 204, as described above. To maximize light transmis(cid:173)
`electrical signals to and from lid 122.
`sion to and from excitation/detection pair 600, the space 25
`Lid 122 includes a secondary processing board 720 that
`between opening 502 and lid heater 204 is advantageously
`facilitates communication between main processing board
`made small during operation.
`702 and stepper motors 302, 316, as well as shuttle 232.
`Excitation light that enters reaction vessel 616 excites the
`Secondary processing board 720 includes connectors 722 for
`fluorescent label or probe therein, which fluoresces, thereby
`cables 212, a connector 724 that connects a cable 726 to
`generating light of a different wavelength. Some of this light 30
`shuttle 232, and connectors 732 and 734 for cables 736, 738
`exits reaction vessel 616 on detection light path 620 and
`that provide control signals to the x and y stepper motors
`passes through opening 502. Beam splitter 612 directs a
`302, 316. Routing paths (not shown) in secondary process(cid:173)
`substantial portion of the fluorescent light through filter 624,
`ing board 720 establish appropriate signal connections
`which filters out the excitation frequency, and lens 626,
`between the various connectors.
`which focuses the light onto the active surface of photodiode 35
`Cable 726 is used to communicate control signals for
`628. Photodiode 628 generates an electrical signal in
`detection module 234, such as activating and deactivating
`response to the incident light. This electrical signal is
`individual light sources, and to receive signals from the
`transmitted by a readout si