USOO7148043B2
`
`(12)
`
`United States Patent
`Kordunsky et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,148,043 B2
`Dec. 12, 2006
`
`(54) SYSTEMS AND METHODS FOR
`FLUORESCIENCE 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
`past Sh used under 35
`
`(21) Appl. No.: 10/431,708
`(22) Filed:
`May 8, 2003
`O
`O
`Prior Publication Data
`US 2004/0224317 A1
`Nov. 11, 2004
`
`(65)
`
`(51) Int. Cl.
`(2006.01)
`CI2P 19/34
`(2006.01)
`CI2O I/68
`(52) U.S. Cl. ......................... 435/91.2, 435/6:435/91.1
`(58) Field of Classification Search ............. 250/4581
`435/6. 91.1, 91.2: 422/82.05 8207
`See application file for complete search history.
`References Cited
`U.S. PATENT DOCUMENTS
`
`(56)
`
`2f1993 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
`5,473,437 A 12/1995 Blumenfeld et al.
`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
`
`1/2000 Woudenberg et al.
`6,015,674. A
`2/2000 Cunanan
`6,024,920 A
`3/2000 Andrews et al.
`6,043,880 A
`6,140,054 A 10/2000 Wittwer et al.
`6,144,448 A 1 1/2000 Mitoma
`6,174,670 B1
`1/2001 Wittwer et al.
`6,197.575 B1
`3, 2001 Gr
`K-1
`riffith et al.
`6,337,435 B1
`1/2002 Chu et al.
`6,359,284 B1* 3/2002 Hayashi et al. .......... 250/458.1
`6,369,893 B1
`4/2002 Christel et al.
`6,569,631 B1* 5/2003 Pantoliano et al. .......... 435/71
`6,818,437 B1
`1 1/2004 Gambini et al.
`2002/0064780 A1* 5/2002 Gold et al. .................... 435/6
`2003/0015668 A1* 1/2003 Montagu ................. 250/458.1
`OTHER PUBLICATIONS
`PCT International Preliminary Report on Patentability for PCT/
`USO4f14566.
`* 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
`ment.
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`18 Claims, 7 Drawing Sheets
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`Agilent Exhibit 1202
`Page 1 of 16
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`U.S. Patent
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`Dec. 12, 2006
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`Sheet 1 of 7
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`Dec. 12, 2006
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`A76, 2
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`U.S. Patent
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`Sheet 3 of 7
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`US 7,148,043 B2
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`is B.
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`CO
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`N2
`N Nat St.
`N St.
`NSee
`NSat
`N N Sde
`N N N NSlee See
`NSat St.
`N St.
`Sde
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`Sde
`Ne See
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`N a Nat Na Sate
`S-at Sot
`Side
`Nat
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`CN R.
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`FIG. 4
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`234
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`234'
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`502
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`so
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`504
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`e 6
`ros
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`FIG. 5A
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`FIG. 5B
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`Sheet 5 of 7
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`Dec. 12, 2006
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`Dec. 12, 2006
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`Sheet 7 Of 7
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`PREPARE REACTION VESSELS
`WITH SAMPLES TO BE ANALYZED
`
`MOUNT DETECTION MODULE
`ON SHUTTLE
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`PLACE REACTION VESSELS IN
`SAMPLE WELLS
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`CLOSE LID AND PLACE UNIT
`INTHERMAL CYCLER BASE
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`CALIBRATE DETECTION MODULE
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`PERFORM PCR CYCLE
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`1.
`SYSTEMIS AND METHODS FOR
`FLUORESCIENCE DETECTION WITH A
`MOVABLE DETECTION MODULE
`
`BACKGROUND OF THE INVENTION
`
`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
`an increasingly time-consuming task, and errors in calibra
`tion may introduce significant errors in Subsequent measure
`mentS.
`In addition, existing fluorometers generally are designed
`such that the light sources and detectors are fixed parts of the
`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
`ferent light source and/or detector. Many existing fluorom
`eters make it difficult for an experimenter to reconfigure
`light sources or detectors, thus limiting the variety of fluo
`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
`maximize the data obtained in an assay, experimenters often
`include multiple fluorescent labeling agents that have dif
`ferent excitation and/or emission wavelengths. Each label
`ing agent is adapted to bind to a different target sequence, in
`principle allowing multiple target sequences to be detected
`in the same sample. Existing fluorometers, however, do not
`facilitate such multiple-label experiments. Many fluorom
`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,
`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,
`slowing the data collection process.
`Therefore, an improved fluorometer for a thermal cycler
`that overcomes these disadvantages would be desirable.
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`The present invention relates in general to fluorescence
`detection systems and in particular to a fluorescence detec
`tion system having a movable excitation/detection module
`for use with a thermal cycler.
`Thermal cyclers 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 cyclers include but are
`not limited to amplification of nucleic acids using proce
`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
`tation) radiation, Such as a light Source of appropriate
`wavelength, thereby exciting the probe and causing fluores
`cence. The emitted radiation is detected using an appropriate
`detector, such as a photodiode, photomultiplier, charge
`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
`excitation light and detects the emitted radiation. The use
`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
`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
`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
`
`BRIEF SUMMARY OF THE INVENTION
`
`Embodiments of the present invention provide fluores
`cence detection in a thermal cycling apparatus. According to
`one aspect of the invention, a fluorescence detection appa
`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
`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
`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
`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
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`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
`ably mounted therein, the detection module including an
`15
`excitation/detection channel, the excitation/detection chan
`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
`tially positioned in optical communication with each of the
`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
`of the nature and advantages of the present invention.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIG. 1 is a perspective view of a thermal cycling appa
`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
`40
`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. 5A-B are bottom views of detection modules
`according to alternative embodiments of the present inven
`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
`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.
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`DETAILED DESCRIPTION OF THE
`INVENTION
`
`An exemplary apparatus embodiment of the present
`invention will be described with reference to the accompa
`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.
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`4
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`I. Exemplary Apparatus
`FIG. 1 is a perspective view of a thermal cycling appa
`ratus 100 according to an embodiment of the present inven
`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
`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
`Engine(R), DyadTM, and TetradTM thermal cyclers sold by MJ
`Research, Inc., assignee of the present application.
`Lid assembly 112 includes a sample unit and a fluores
`cence detection apparatus, disposed within a lid 122; these
`components will be described below. Lid 122 has a handle
`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.
`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
`as Microseal. RB (made by MJ Research, Inc.), wax products
`such as Chill-outTM (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
`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
`ments, electrical connection elements for connecting the
`heating elements to base unit 110, and mechanical connec
`tion elements. These components (not shown) may be of
`conventional design. Sample unit 202 also provides electri
`cal connections for lid heater 204 and fluorometer assembly
`206 via multiwire cables 212, which are detachably con
`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
`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
`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
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`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
`imide (PEI). Neutral-density or other types of filters may be
`placed over the fluorescent material in order to avoid satu
`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
`geously minimized. Where multiple calibration elements
`222 are provided, different materials may be used for
`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
`tional design. Examples of Suitable designs include sample
`unit and lid heater components of the various AlphaTM
`modules sold by MJ Research, Inc., assignee of the present
`application.
`Fluorometer assembly 206 includes a support frame or
`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
`204; one of skill in the art will appreciate that this arrange
`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
`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
`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
`operates to turn lead screw 318, thereby moving shuttle 232
`along the y direction (indicated by arrow). Limit Switches
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`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
`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
`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.
`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,
`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
`tion module. In one embodiment, connectors 330 provide
`mounting for a cylindrical member (not shown) that pivot
`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
`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
`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. 5A is a bottom view of one embodiment of detection
`module 234, showing four openings 502,504,506, 508 for
`four independently controlled fluorescent excitation/detec
`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 502, 504, 506, 508 corre
`sponds to the spacing of sample wells 210. Thus, when
`opening 502 is placed in optical communication with one of
`the sample wells 210, openings 504, 506, and 508 are each
`in optical communication with a different one of the sample
`wells 210. Openings 502,504,506, 508 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
`transparency to the excitation and detection light wave
`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 512, 514, 516, 518 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
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`US 7,148,043 B2
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`more instances of excitation/detection pair 600, each of
`which provides an independent fluorescence detection chan
`nel. Excitation/detection pair 600 is arranged inside opaque
`walls 602, which provide optical isolation from other exci
`tation/detection pairs that may be included in detection
`module 234, as well as from external light sources. An
`excitation light path 604 includes a light-emitting diode
`(LED) or other light source 606, a filter 608, a lens 610, and
`a beam splitter 612. A detection light path 620 includes beam
`splitter 612, a filter 624, a lens 626, and a photodiode or
`other photodetector 628. Beam splitter 612 is advanta
`geously selected to be highly transparent to light of the
`excitation wavelength and highly reflective of light at the
`detection (fluorescent response) wavelength.
`The components of excitation light path 604 are arranged
`to direct excitation light of a desired wavelength into a
`reaction vessel 616 held in a sample well 210 of sample
`block 202. The desired wavelength depends on the particular
`fluorescent labeling agents included in reaction vessel 616
`and is controlled by selection of an appropriate LED 606 and
`filter 608. Optical communication between the excitation/
`detection pair 600 and reaction vessel 616 is provided by
`opening 502 in opaque walls 602 and a hole 220 through lid
`heater 204, as described above. To maximize light transmis
`sion to and from excitation/detection pair 600, the space
`between opening 502 and lid heater 204 is advantageously
`made Small during operation.
`Excitation light that enters reaction vessel 616 excites the
`fluorescent label or probe therein, which fluoresces, thereby
`generating light of a different wavelength. Some of this light
`exits reaction vessel 616 on detection light path 620 and
`passes through opening 502. Beam splitter 612 directs a
`substantial portion of the fluorescent light through filter 624,
`which filters out the excitation frequency, and lens 626,
`which focuses the light onto the active surface of photodiode
`628. Photodiode 628 generates an electrical signal in
`response to the incident light. This electrical signal is
`transmitted by a readout signal path 630 to circuit board 634,
`which routes the signal to electrical connector 424 for
`readout. Circuit board 634 and/or signal path 630 may also
`include other components, such as pre-amplifiers, for shap
`ing and refining the electrical signal from photodiode 628.
`LED 606 and photodiode 628 may be controlled by
`signals received via connector 424, as indicated by respec
`tive control signal paths 636, 638. Control signals for LED
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`606 may operate to activate and deactivate LED 606 at
`desired times; control signals for photodiode 628 may
`operate to activate and deactivate photodiode 628 at desired
`times, adjust again parameter, and so on.
`While FIG. 6 shows one excitation/detection pair 600, it
`is to be understood that an embodiment of detection module
`234 may contain any number of such pairs, each of which is
`advantageously in optical isolation from the others and has
`its own opening for optical communication with the sample
`wells (e.g., openings 504,506, 508 of FIG. 5). The various
`excitation/detection pairs are independently controlled and
`independently read out, but their respective control and
`readout paths may all be coupled to circuit board 634.
`The configuration of excitation/detection pairs may be
`varied from that shown, and the excitation and detection
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`light paths may include additional components, fewer com
`ponents, or any combination of desired components. The
`optics may be modified as appropriate for a particular
`application (e.g., the optical path may be shorter in embodi
`ments where lid heater 204 is not included) and use any
`number and combination of components including but not
`limited to lenses, beam splitters, mirrors, and filters. While
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`LEDs provide a compact and reliable light source, use of
`other types of coherent or incoherent light sources, such as
`laser diodes, flash lamps, and so on, is not precluded.
`Similarly, the detectors are not limited to photodiodes; any
`type of photodetector may be substituted, including photo
`multipliers and charge-coupled devices (CCDs). Each exci
`tation/detection pair is advantageously configured as a self
`contained assembly, requiring only external electrical
`connections to make it operational. Because the length of the
`excitation and detection optical paths do not vary from one
`experiment to the next, it is desirable to fixedly mount and
`optimize the various optical components of each excitation/
`detection pair 600 inside detection module 234 during
`manufacture so that further adjustments during operation are
`not required.
`FIG. 7 is a block diagram illustrating electrical connec
`tions for lid assembly 112. A main processing board 702 is
`mounted in lid assembly 112. Main processing board 702
`includes a primary signal processor 704, a stepper motor
`driver unit 706, a connection 708 for electrical power, and a
`connection 710 for an external computer (e.g., a personal
`computer, or PC). Main processing board 702 also provides
`connectors 214 for cables 212 that provide transmission of
`electrical signals to and from lid 122.
`Lid 122 includes a secondary processing board 720 that
`facilitates communication between main processing board
`702 and stepper motors 302, 316, as well as shuttle 232.
`Secondary processing board 720 includes connectors 722 for
`cables 212, a connector 724 that connects a cable 726 to
`shuttle 232, and connectors 732 and 734 for cables 736, 738
`that provide control signals to the X and y stepper motors
`302,316. Routing paths (not shown) in secondary process
`ing board 720 establish appropriate signal connections
`between the various connectors.
`Cable 726 is u