(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`(19) World Intellectual Property Organization
`International Bureau
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`I lllll llllllll II llllll lllll llll I II Ill lllll lllll 111111111111111111111111111111111
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`(43) International Publication Date
`17 May 2001 (17.05.2001)
`
`PCT
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`(10) International Publication Number
`WO 01/35079 Al
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`(51) International Patent Classification7:
`21/25, BOIL 7/00
`
`GOIN 21164,
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`(21) International Application Number: PCT/US00/30771
`
`(22) International Filing Date:
`9 November 2000 (09 .11.2000)
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, Fl, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/165,267
`
`12 November 1999 (12.11.1999) US
`
`English
`
`English
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): E. I. DU
`PONT DE NEMOURS AND COMPANY [US/US]; 1007
`Market Street, Wilmington, DE 19898 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): LEE, Jerald, D.
`[US/US]; 857 Burrows Run Road, Mendenhall, PA 19357
`(US). DABELL, Stanley, D. [US/US]; 2 Memory Lane,
`Newark, DE 19702 (US).
`
`(74) Agent: LI, Kening; E.I. Du Pont de Nemours and Com(cid:173)
`pany, Legal Patent Records Center, 1007 Market Street,
`Wilmington, DE 19898 (US).
`
`Published:
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments.
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`
`(54) Title: FLUOROMETER WITH LOW HEAT-GENERATING LIGHT SOURCE
`
`(57) Abstract: This invention concerns a fluorometer preferably combined with a
`thermal cycler useful in biochemical protocols such as polymerase chain reaction
`(PCR) and DNA melting curve analysis. The present fluorometer features a low
`heat-generating light source such as a light emitting diode (LED), having a one-to-one
`correspondence to each of a plurality of sample containers, such as capped PCR tubes
`in a standard titer tray. The fluorometer of the present invention further comprises
`an optical path between each LED and its correspondingly positioned container, and
`another optical path between each fluorescing sample within the positioned container
`and an optical signal sensing means. The instrument can be computer controlled.
`
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`TITLE
`FLUOROMETER WITH LOW HEAT-GENERA TING LIGHT SOURCE
`FIELD OF THE INVENTION
`This invention relates to instrumentation, particularly to instruments for detecting and
`5 measuring fluorescence, and more particularly to fluorescence measurements usable in
`conjunction with a variety of applications including use in assays based on polymerase chain
`reaction (PCR).
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`BACKGROUND OF THE INVENTION
`Many reactions are characterized by the occurrence, or changes in level, of
`fluorescence when illuminated by a suitable excitation wavelength. In these types of
`reactions, a fluorescent sample absorbs light of a given wavelength and, in response thereto,
`emits light of a different wavelength.
`Fluorescence may be inherent in the involved reagents or it may be provided
`deliberately by a suitable marker incorporated in the reactants. Hence, instruments for
`15 measuring fluorescence, fluorometers, are commonplace in the laboratory environment.
`Several difficulties exist with many stand-alone fluorometers and those combined
`with other instrumentation. First, it is difficult to obtain very high intensity light in the
`proper wavelength from instruments which utilize a halogen or laser unitary light source
`without generating a large amount of heat. Similarly, since tungsten lights and the like must
`be on continuously to reach and operate under stable conditions, they also generate a large
`amount of heat. This large amount of heat can shorten the life of the lamp and should be
`dissipated because it may heat up the sample, thereby changing its fluorescent light emitting
`characteristics. Thus, these types of instruments require extensive cooling for the light
`source, and such light sources require frequent replacement.
`In addition, for those instruments in which a group of samples may be tested
`simultaneously, a great deal of excitation light energy can be lost through diversion between
`the samples. This translates into a lower excitation efficiency.
`Other fluorometers either lack sufficient sensitivity or are so expensive in
`construction as to be impractical for many purposes. For example, many fluorometers with a
`halogen light source do not have adequate sensitivity. This type offluorometer has a limited
`dynamic range since all samples are illuminated and imaged at the same time if a typical
`charge coupled device (CCD) type camera is used. A laser light source type fluorometer can
`have better performance, but the laser light source is more expensive.
`Many protocols, particularly in the broad field of microbiology, require repetitive,
`controlled temperature regimes. Apparatus filling this need are called "thermal cyclers". It
`is useful to combine fluorometers with thermal cyclers for facilitating receipt of results
`which depend on fluorescence measurements to indicate reactions. One protocol which
`utilizes a thermal cycler is the polymerase chain reaction (PCR) (see U.S. Patent 4,683,202
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`(Mullis)). To determine if amplification has occurred at the end of PCR, fluorescent dyes
`may be used as indicators, particularly intercalating dyes that fluoresce when bound to
`double stranded DNA but do not bind, or bind very inefficiently to single strands and have
`no or little signal in the presence of single strands.
`Certain commercially available fluorometer/thermal cyclers can accommodate a
`standard ninety-six well tray of reaction tubes and include a fluorometer which uses a single,
`powerful incandescent light source to project light through an optical system to illuminate
`the tray and excite the fluorescent dyes therein to indicate positive reactions. Other
`commercially available instruments use a laser instead of an incandescent light source and a
`IO mechanical scanning device to isolate the signal from a reaction tube via a fiber optic cable
`onto a photodiode array.
`Fig. 1 depicts a schematic of a system similar to certain commercially available
`instruments. It uses a single incandescent light source, lamp 1, which is a halogen projection
`lamp. Cooling is provided by a fan not shown. The light output passes through shutter 2
`15 which is actuated by means not shown to shield the system when measurements are not
`being made. The light is directed toward the entirety of the sample tubes, not shown, held in
`sample plate 10 which is in intimate contact with a heating/cooling block 3 and in
`coordination with a data accumulation system not shown. Standard plates, or "trays", used
`in this system hold 96 tubes. The directed beam of light passes through an excitation filter 4.
`The filtered light from filter 4 passes rectangular aperture 5 to confine the light to the sample
`tray area. Beamsplitter 6 reflects the light toward beam folding mirror 7. The light is then
`directed to fresnel lens 8. Fresnel lens 8 directs the light onto individual lenses mounted in
`plate 9, one lens per sample tube carried by plate 10. Once the light contacts the sample
`material any emitted light passes through beam splitter 6 and then through emission filter 11,
`lens 12, and into CCD type camera 13. CCD type camera 13 acquires an image of the entire
`sample tray. A computer program is used to calculate the fluorescent intensity of each
`sample tube from the image. The power measured by CCD type camera 13 indicates the
`reaction in the individual tube.
`Other commercially available systems use an argon ion laser as the light source. In
`these systems, light from the argon ion laser passes through a dichroic mirror, a lens and a
`multiplexer which provides a fiber optic cable for each well of a 96-well plate. Excited light
`returns to the mirror and is reflected into a spectrograph which separates the light into a
`pattern that falls on a linear CCD detector. Appropriate filters are included in the optical
`paths.
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`Another commercially available system is partially depicted schematically in
`Figure 1 a. This instrument combines a "microvolume fluorometer" with thermal cycler 120.
`The light source in this instrument is a single light emitting diode (LED) 100 which projects
`light through lens and filter 102 to dichroic mirror 103 to reading lens 104 and then to the
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`bottom of reaction tube 106. The reaction tubes, thirty-six in number, are held in carousel
`108 which is stepped by motor 110 to place each tube in sequence over the reading lens 104.
`Any resultant emission reflects from mirror 103 and from one of the dichroic, color selective
`mirrors 114 through lenses 116 to one of photohybrids 118. A signal then is processed in an
`associated computer and printer not shown. The cycler 120 is air-cooled and heated using
`fan motor and associated fan blades 112 and heater coil 122 driving heated or cold air as
`directed by the program within the computer. Motor 124 positions the optical system as
`required. This system is not suited to a standard 8 by 12 tray and is expensive because it
`requires complex positioning mechanisms to present the tubes to the fluorometry system.
`In view of the problems discussed above, there is a need to provide an inexpensive
`fluorometer having either a simple positioning mechanism or no
`positioning mechanism. In addition, there is a need for a fluorometer characterized by low
`heat-generating light sources using minimal power and which eliminates waste of excitation
`energy. There is also a need to provide light sources with rapid stabilization. Further, there
`exists a need for a highly sensitive fluorometer with an improved signal to noise ratio.
`SUMMARY OF THE INVENTION
`The present invention concerns a fluorometer, comprising:
`plurality of low heat-generating light sources; means for positioning a plurality of
`containers for containing potentially fluorescing sample into optical communication with
`said light sources, wherein each light source corresponds with one of said containers when
`said container is in position; a first optical path means for directing light from said light
`source to said corresponding container; optionally an excitation filter in said first optical path
`means for allowing transmission therethrough of an excitation wavelength from the light
`generated from each light source; an optical signal sensing means in optical communication
`with any fluorescing sample in said positioned containers; a second optical path means for
`directing emitted light from any fluorescing sample to said optical signal sensing means; and
`optionally an emission filter in said second optical path for allowing transmission
`therethrough of emitted light from any fluorescing sample and for substantially blocking
`transmission of light of wavelengths other than the wavelengths of said emitted light.
`The present invention also concerns a combined fluorometer and thermal cycler,
`comprising the fluorometer described above, wherein the containers are sample tubes, in
`combination with a thermal cycler, said thermal cycler comprising: a thermally controlled
`base having a plurality of wells, each well capable of holding a capped sample tube, or the
`tube portions of an integral tube/holder, in close contact; a thermally controlled cover having
`a plurality of apertures corresponding to each sample tube, said cover in operative condition
`mechanically biasing the cap of each said sample tube into said close contact, each said
`aperture expanding outward from said cap; and programmable control means for controlling
`the temperature of said sample tubes according to a selected protocol.
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`The present invention further concerns a method for detecting a fluorescence signal
`from polymerase chain reaction amplified material, comprising the steps of: positioning the
`material into optical communication with the light sources of the fluorometer of the present
`invention described above; exposing the material to an excitation wavelength; detecting the
`emitted light with the optical signal sensing means; and comparing a differential of the
`emitted light level to a pre-determined reference level.
`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure 1 is a schematic drawing of an optical system of the prior art.
`Figure la is a schematic drawing of an optical system of the prior art along with a
`thermal cycling system.
`Figure 2 is a schematic drawing of one embodiment of the present invention.
`Figure 3 is a schematic drawing of an off-axis embodiment of the present invention.
`Figure 4 is a schematic drawing an off-axis of another embodiment of the present
`invention.
`Figure 5 is a schematic drawing of yet another embodiment of the present invention.
`Figure 6 is a side elevational view of a thermal cycler in combination with a
`fluorometer of the present invention.
`Figure 7 is a schematic view of elements of the combined thermal cycler and
`fluorometer of Fig. 6 as seen by partially breaking away the near side of the covers thereof.
`Figure 8 is a cross-sectional view of a sample tube held in a temperature controlled
`well with a heating cover.
`Figure 9a is an electrical schematic showing feedback control of a light source.
`Figure 9b is an electrical block diagram of the LED selection circuitry.
`Figure 9c is an electrical block diagram of one embodiment for the programmable
`control means for the present invention.
`Figure 10 is a partial elevational schematic view of one embodiment for lens
`arrangement for the present invention.
`Figure 11 is a schematic view of an optical arrangement for the second optical path
`means of the present invention.
`Figure 12 shows an embodiment of the heating cover shown in Figure 8 of the
`present invention.
`Figure 13 is a schematic diagram showing elements of the present invention
`including a dichroic mirror.
`Figure 14 is a schematic diagram of another embodiment of the present invention.
`Figures l 5a and l 5b are schematic diagrams of a further embodiment of elements of
`the present invention.
`Figure 16 is a schematic view of another preferred embodiment of the present
`invention showing the use of different containers and holder.
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`Figure 17 is an enlarged sectional view of the area denoted "A" in Fig. 16.
`Figure 18a and 18b are schematics showing one embodiment for compensating for
`changes in light source output where Fig. 18b is the electrical schematic for the component
`layout of Figure 18a.
`Figure 18c is a schematic of one embodiment of programmable control means of the
`present invention.
`Figure 19 is a plane view of a "pixelated" fresnel lens useful in the present
`fluorometer.
`Figure 20 is an elevational, sectional view of one embodiment for mounting the light
`sources of the present invention.
`Figure 21 is a perspective view of a preferred embodiment of the combined thermal
`cycler and fluorometer of the invention shown with associated desktop computer.
`DETAILED DESCRIPTION OF THE INVENTION
`To circumvent the difficulties described above, the fluorometer of the present
`invention preferably uses a plurality of light emitting diodes (LEDs) as the light sources. By
`using LEDs, a large amount of heat is not generated, thereby minimizing sample heating,
`easing heat dissipation problems, and extending the life of the light source. In addition, by
`providing a one-to-one correspondence of light source to sample container with the directed
`light falling inside the container, waste of excitation energy is largely eliminated allowing
`for the use of a light source that uses minimal power.
`In addition to the advantageous arrangement of components of the first and second
`optical paths means. the present invention also has the benefit that for the plurality of
`potentially fluorescing samples, there is an equal number of low heat-generating light
`sources, and the output of any one of the light sources is dedicated to a particular,
`correspondingly positioned container containing the potentially fluorescing samples in an
`optical system in which the positioning means, such as a sample tube tray, can be viewed by
`an optical signal sensing means.
`The present invention concerns a fluorometer, comprising a plurality of low heat(cid:173)
`generating light sources; means for positioning a plurality of containers for containing
`potentially fluorescing sample into optical communication with the plurality of light sources,
`wherein each light source corresponds with one of said containers when said container is in
`position; a first optical path means for guiding light from said light source to said
`corresponding container; an excitation filter in said first optical path means for allowing
`transmission therethrough of an excitation wavelength from light generated from each light
`source; an optical signal sensing means in optical communication with any fluorescing
`sample in said positioned containers; a second optical path means for directing emitted light
`from any fluorescing sample to said optical signal sensing means; and an emission filter in
`said second optical path for allowing transmission therethrough of emitted light from any
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`fluorescing sample and for substantially blocking transmission of light of wavelengths other
`than the wavelengths of said emitted light.
`As shown in Fig. 2, the fluorometer of the present invention includes a plurality of
`low heat-generating light sources 20. By "low heat-generating" is meant below the level at
`5 which active cooling of the light source, such as via a fan, is required. Such low heat-
`generating light sources generally use only low or minimal power for energizing. The light
`sources used are capable of emitting a wavelength that can excite an indicator in a sample.
`Each light source is in optical communication with the potentially fluorescing sample when
`the container containing such sample and with which such light source corresponds, is
`placed in position in the fluorometer where such optical communication can occur. Suitable
`light sources for use in the present invention are solid state light sources including laser
`diodes and light emitting diodes. A preferred light source is a light emitting diode (LED).
`In a preferred embodiment of the present invention , the LEDs are blue LEDs. NSPB500S
`LEDs are available from Nichia Corp. (3775 Hempland Road, Mountville, PA 17554) and
`are suitable LEDs for the present invention. Blue LEDs are preferred because the preferred
`intercalating dye, SYBR Green (available from Molecular Probes, Inc., 4849 Pitchford Ave.
`Eugene, OR 97402), is sensitive to the exciting wavelength of blue. If other fluorescing
`materials are used in the sample material, it is a simple matter to switch to an alternative
`LED light source color and excitation filter that will excite a given dye.
`The low heat-generating light sources of the present invention can provide adequate
`power to the potentially fluorescing sample contained in the light sources' corresponding
`positioned containers because in the present invention light is not wasted on the spaces in
`between the positioned containers. One low heat-generating light source is provided for
`each of the containers in a one-to-one correspondence. There is an array of low heat-
`generating light sources. A representative example of the array of low heat-generating light
`sources 20 can be seen in Fig. 2.
`As shown in Figs. 2, 3, 4, 5, 10, 13, 14, 15a, 15b, and 20, light source holder 21 can
`be used for mounting or carrying light sources 20. As shown in Fig. 7 for a combined
`fluorometer and thermal cycler of the present invention described in more detail below, a
`light source support plate 221 can be used to carry the light sources 20, such as an array of
`LEDs. The light sources can be held or mounted in such holders or support plates in drilled
`cavities and can be held with cement or other suitable adhesive after careful aiming toward a
`particular optic in the first optical path means, shown for example in Fig. 7 as the center of
`lens 38. In Fig. 20 another embodiment for a means for mounting the light sources is shown.
`Light sources 20, preferably LEDs, can be press fit into holes 23 in light source holder 21.
`This places the inner part of flanges 66, which are smooth and fairly perpendicular to the
`axis of light sources 20 against the surface of light source holder 21. A sheet of soft rubber
`or other elastomer 60 covers the other side of light sources 20 and is perforated so that two
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`electrical leads, not shown, on each light source 20 can pass through and enter holes in
`circuit board 62. This assembly can be held under pressure by fasteners 64. To prepare this
`assembly, sheet 60 is placed on top of circuit board 62, which may be part of a computer,
`such as a PC. The leads of light sources 20 are inserted into circuit board 62 through sheet
`60 and circuit board 62 is placed over light source holder 21 so that each light source 20 is in
`its hole 23. Fasteners 64 are inserted and torqued. The leads can then be soldered to circuit
`board 62. This embodiment firmly mounts light sources 20. Proper mounting of light
`source holder 21 assures aiming oflight sources 20.
`The mounting for the low heat-generating light sources can be adjusted to provide
`any reasonable ratio of spacing between each light source desired to suit design
`considerations. Preferably, a symmetrical spacing arrangement for both the low heat(cid:173)
`generating light sources and their corresponding containers is used. The distances between
`each light source can be equal. This equal distance, however, is only a matter of
`convenience and suits the commercially available 96 sample tube holders that can be used as
`positioning means in the present fluorometer.
`The present fluorometer can further comprise means for powering each of the low
`heat-generating light sources (see, for example, Fig. 9c). Each light source can be energized
`simply by attaching it to a power source. Alternatively, each light source can be powered in
`a selected sequence by programmable control means, such as a computer. Powering means
`can be capable of powering each light source sequentially, simultaneously, randomly, or any
`combination thereof. If there are a fewer number of containers than the number of light
`sources, this can be easily accommodated through programmable control means, whereby
`either the light source corresponding to a position within the positioning means without a
`container may not be powered and/or have its optical signal sensed. Another method of
`driving an array of LEDs is to use a crosspoint switch arrangement. The anodes of each
`column of LEDs are wired in parallel. The cathodes of each row of LEDs are also wired in
`parallel, A single LED is illuminated by selecting the desired row and column. Column
`signals may be switched by PNP transistors connected to a positive supply voltage. Row
`signals may be switched by NPN transistors connected to ground.
`Figure 9b shows three thirty-two channel serial-to-parallel conveners 240 that can
`form half of the electrical drive circuitry that can be used to power the LEDs of one
`embodiment of the present invention. HV51s with open drain outputs available from
`Supertex, Inc. (1235 Bordeaux Drive, Sunnyvale, CA 94089) can be used as such converters.
`One of these channels is shown in Fig. 9a as 238 which shows the other half of the LED
`drive circuitry. In the circuitry shown, digital-to-analog (D/ A) converter 232, such as a L TC
`1446 by Linear Technology Corporation (15 Research Place, North Chelmsford, MA
`01863), provides an output of 0 to 5 volts. Summing amplifier 234, such as an LM2904 by
`National Semiconductor Corporation (2900 Semiconductor Drive, Santa Clara, CA 95051 ),
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`compares the DIA output to that of the output of trans-impedance amplifier 236, such as a
`CA3140 by Intersil, Corp. (2401 Palm Bay Road, N.E., Palm Bay, FL 32905), which is
`driven by optional detector 25, such as a silicon photodiode VTD34 available from
`PerkinElmer Analytical Instruments (761 Main Avenue, Norwalk, CT 06859). Summing
`amplifier 234 causes these two voltages to be equal in magnitude. Detector 25 and trans(cid:173)
`impedance amplifier 236 should be arranged to insure that the output voltages from DI A
`converter 232 and trans-impedance amplifier 236 are opposite in sign. In this embodiment,
`the light intensity of an LED light source is pinned to a reference voltage. This reference
`voltage can be set by programmable control means 500, such as a computer, via DI A
`converter 232 to uniformize the output of the plurality of light sources.
`As mentioned above, the present fluorometer may further comprise means for
`uniformizing the output from the low heat-generating light sources. By "uniformizing the
`output" is meant that the intensity of each light source is measured and adjusted, if
`necessary, to ensure that the intensity of light from each light source is equal to that of the
`other light sources in the fluorometer. A variation in the light beam intensity sensed by the
`uniformizing means is used to correspondingly increase or decrease the voltage of the power
`source for the light source so as to maintain the light beam intensity constant and at a
`predetermined level. Consequently, variations in the light beam intensity due to fluctuations
`in the power supply voltage, the age of the light source and the like are prevented from
`affecting the fluorescent emissions by the particles in the sample being tested.
`One embodiment of a uniformizing means is shown in Fig. 3 as detector 25 and one
`embodiment for associated circuitry is shown in Figs. 9a and 9b, as was discussed above. A
`preferred uniformizing means is a silicon photodiode and its associated feed-back circuitry.
`The uniformizing means is preferably located in a position where the light from each of the
`low heat-generating light sources can fall upon it. For example, in Fig. 3, such a location is
`adjacent to excitation filter 42 between meniscus lenses 38, 40, or on the far side of
`excitation filter 42 from light sources 20. Another suitable location can be in the center of
`excitation filter 42, as shown. A relatively small uniformizing means is preferred so the light
`going toward the positioned containers is not excessively blocked.
`Quantitatively, the fluorescent emissions of the excited sample in the container are a
`function of the intensity of the excitation light beam from light source and the concentration
`of fluorescent particles in the sample, or the total number of fluorescent particles in the
`sample cell. Thus, any fluctuation in the intensity of the excitation light beam would result
`in a corresponding change in the intensity of the fluorescent emissions and cause an error
`35 which is a function of the intensity change in the excitation beam.
`Because the LEDs 20 are operated at a nominally high current, there is not much
`room for a further increase in current should the feedback circuit described previously call
`for more. Another approach, therefore, indicated schematically in Fig. 18a and appropriate
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`circuitry in Fig. 18b, is to supply the LEDs with an unvarying current and to form a ratio
`between the sample signal measured by optical signal sensing means 36 and the signal
`produced by at least one silicon photodiode 25 monitoring the LED output. In this way
`variations in sample signal are compensated as the LED output changes.
`Another technique for uniformizing the output of the light sources is to allow the
`array of LEDs to excite a calibration phosphor. The calibration phosphor can be a sheet of
`plastic that fluoresces, such as the UL TEM® type polyetherimide, available from AIN
`Plastics, Inc. (249 E. Sandford Blvd, Mount Vernon, NY 10550), which can withstand
`temperatures as high as 200°C. The intensity oflight emitted for each source is recorded and
`normalized using the detected signals from the phosphor.
`The fluorometer of the present invention includes means for positioning a plurality of
`containers for containing potentially fluorescing sample into optical communication with
`said light sources, wherein each light source corresponds with one of said containers when in
`position. Such positioning means include a sample plate, a titer plate, a sample holder, a
`sample tray, a carriage, or other transport device known by those of skill in the art as capable
`of holding or accommodating a plurality of containers for containing potentially fluorescing
`samples.
`The positioning means containing the containers is mounted or positioned in the
`fluorometer in such a manner as to place the containers (and likewise the potentially
`fluorescing samples) into optical communication with the corresponding light source.
`The containers can include containers capable of being separated from the
`positioning means or can be integrally formed within the positioning means. Alternatively,
`the containers can be wells formed within the positioning means that are capable of holding
`the potentially fluorescing sample. Each embodiment will be of a size that will fit the
`particular configuration of the fluorometer. Preferably, the containers are sample tubes.
`Sample tubes suitable for the present invention are commercially available. Containers 32
`are shown in Fig. 2 as being held in positioning means which is shown as sample plate 30
`which can be a titer plate. Preferably, there is an array of containers held by the positioning
`means. Most preferably, the positioning means and containers together are an array of
`8 sample tubes by 12 sample tubes to total 96 held in a sample plate. The containers can be
`capped or covered by other means known in the art. As used herein "cap" includes caps and
`other covers for sealing containers.
`The present fluorometer includes a first optical path means for directing light from
`said plurality of low heat-generating light sources to a potentially fluorescing sample in the
`corresponding positioned container. Thus, said first optical path means optically connects
`each low heat-generating light source to its corresponding positioned container via one of a
`variety of optical arrangements. The first optical path means can include a variety of optics
`of conventional construction. The particular arrangement of the optical components along
`9
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`THERMO FISHER EX. 1016
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`WO 01/35079
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`PCT/US00/30771
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`5
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`IO
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`15
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`20
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`25
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`30
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`35
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`the first optical path can be adjusted to provide any reasonable ratio of spacing desired to suit
`design considerations. Suitable optics for guiding light can include at least one of the
`following: a lens, including a condensing lens, an objective lens