(12) United States Patent
`Hayashi et al.
`
`US006515743B1
`US 6,515,743 B1
`Feb. 4, 2003
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`(54) SCANNER-TYPE FLUORESCENCE
`DETECTION APPARATUS USING SMALL
`SIZED EXCITATION LIGHT SOURCE
`
`(75) Inventors: Toshinori Hayashi, KanagaWa (JP);
`Yoshifumi Kurihara, KanagaWa (JP);
`Takahiko Ishiguro, KanagaWa (JP)
`
`(73) Assignee: Tosoh Corporation, Yamaguchi (JP)
`
`5,178,833 A
`
`1/1993 Covain
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`GB
`JP
`
`0 985 927 A2
`1 024 355 A1
`2 000 284 A
`2000-88752
`
`3/2000
`8/2000
`1/1979
`3/2000
`
`........ .. G01N/21/64
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 222 days.
`
`Primary Examiner—F. L. Evans
`(74) Attorney, Agent, or Firm—Sughrue Mion, PLLC
`
`(57)
`
`ABSTRACT
`
`(21) Appl. No.: 09/667,722
`(22) Filed:
`Sep. 22, 2000
`(30)
`Foreign Application Priority Data
`Sep. 22, 1999
`
`(JP) ......................................... .. 11-268893
`
`(51) Int. Cl.7 .............................................. .. G01N 21/64
`(52) US. Cl. ..................... .. 356/317; 356/318; 356/417;
`250/458.1
`(58) Field of Search ............................... .. 356/317, 318,
`356/417; 250/4581, 459.1, 461.1, 461.2
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,406,547 A
`
`9/1983 Aihara
`
`A ?uorescence detection apparatus comprising: a sample
`holder for ?xing and holding sample vessels on a circular
`arc; a partition plate being joined to drive means for rotation
`on the center of the circular arc; an excitation light source;
`excitation light optical means; and ?uorescence optical
`means containing a light guide being ?xed on the partition
`plate for rotation integrally and a photosensor. The photo
`sensor is mechanically discontinued from the drive means
`and ?xedly placed. The ?uorescence signal emission end of
`the light guide is placed facing the photosensor on the
`rotation center axis and the partition plate and the parts ?xed
`thereto are rotated integrally, Whereby ?uorescence detec
`tion of the samples arranged on the circular arc is repeated
`in order.
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`8 Claims, 10 Drawing Sheets
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 1 0f 10
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`US 6,515,743 B1
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`FIG. 1B
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 2 0f 10
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`US 6,515,743 B1
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`FIG. 2
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 3 0f 10
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`US 6,515,743 B1
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`FIG. 3
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`U.S. Patent
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`Sheet 4 0f 10
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`Feb. 4, 2003
`FIG. 4A
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`US 6,515,743 B1
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`FIG. 45
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 5 0f 10
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`US 6,515,743 B1
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`FIG. 5B
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 6 0f 10
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`US 6,515,743 B1
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`FIG. 6A
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`INTERFERENCE
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 7 0f 10
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`US 6,515,743 B1
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`FIG. 7A
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`ELECT
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 8 0f 10
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`US 6,515,743 B1
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`FIG. 8
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 9 0f 10
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`US 6,515,743 B1
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`FIG. 95
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`U.S. Patent
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`Feb. 4, 2003
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`Sheet 10 0f 10
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`US 6,515,743 B1
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`FIG. 10
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`HEAT INSULATION
`MATERIAL
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`US 6,515,743 B1
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`1
`SCANNER-TYPE FLUORESCENCE
`DETECTION APPARATUS USING SMALL
`SIZED EXCITATION LIGHT SOURCE
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`This invention relates to a ?uorescence detection appara
`tus for detecting a ?uorescence signal from a speci?c
`substance contained in a sample and quantifying the speci?c
`substance from the detected ?uorescence signal amount and
`in particular to a ?uorescence detection apparatus useful for
`monitoring a large number of samples in real time (tracing
`change of the ?uorescence signal amount With time) in a
`clinical diagnosis ?eld requiring incubation at a predeter
`mined temperature, such as an enZyme reaction.
`2. Description of the Related Art
`To monitor producing a ?uorescent reaction product by an
`enZyme reaction in real time, etc., it is necessary to detect
`?uorescence While incubating a sample (reaction liquid) at a
`predetermined temperature. Moreover, a large number of
`samples need also to be treated promptly at the same time in
`?elds of clinical diagnosis, etc.
`A?rst method used in the conventional clinical diagnosis
`?eld, etc., is a method of detecting ?uorescence in order
`While transporting samples along a temperature-adjusted
`guide. For example, the temperature of a guide manufac
`tured With a material having good thermal conductivity such
`as an aluminum alloy is adjusted by a heater, etc., samples
`placed in a holder are transported along the guide using a
`chain, a turn table, or the like one or more than one at a time,
`and a ?uorescence signal is detected in order by a ?uores
`cence detector placed along the guide.
`In addition, a second method of detecting ?uorescence at
`the same time about a large number of samples, for example,
`by placing a joint-type sample vessel, titer plate, etc.,
`capable of storing a large number of samples on temperature
`adjustment means is also knoWn. A ?uorescence detection
`apparatus used for the purpose comprises (a) a plurality of
`photosensors or (b) a multichannel-type photosensor or has
`(c) mechanical move means for moving photosensor or light
`guide (means for guiding a ?uorescence signal emitted from
`a sample vessel to photosensor, such as an optical ?ber).
`The apparatus (a) is a ?uorescence detection apparatus for
`using as many photosensors as samples for detecting ?uo
`rescence at the same time to detect a ?uorescence signal
`separately from each sample. In such an apparatus, it is
`common practice to use a light guide for dividing excitation
`light from a light source and guiding to each sample.
`The apparatus (b) is a ?uorescence detection apparatus for
`using an image sensor such as CCD or a photodiode array in
`place of a plurality of photosensors, thereby detecting ?uo
`rescence signals from aligned samples as an image in a state
`in Which the positional relationship betWeen light emission
`points is held. In such an apparatus, it is also common
`practice to guide excitation light from a light source to each
`sample by using a division-type light guide, such as an
`optical device or an optical ?ber.
`In the apparatus (c), the photosensor is moved mechani
`cally on a large number of samples or samples are moved to
`the ?uorescence detection position of the photosensor in
`order; most used is a con?guration of moving the light guide
`mechanically. In this con?guration, an excitation light guide
`and a ?uorescence light guide are used and the ends of both
`guides placed on the sample side are made integral With each
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`other, then both guides are moved at the same time, Whereby
`?uorescence is detected While a large number of samples are
`excited in order.
`To use the ?uorescence detection apparatus in the con
`ventional arts to monitor change of a ?uorescence signal
`With time from a speci?c substance contained in a sample in
`real time While incubating the sample at a predetermined
`temperature, the folloWing problems are involved:
`The ?rst method described above involves the risk of
`insufficient temperature adjustment accuracy, the limit of the
`number of treated samples, carry-over, etc., because samples
`are transported along the temperature-adjusted guide and
`?uorescence is detected in order. That is, it is dif?cult to
`adjust the Whole sample transport guide at a uniform tem
`perature and hold the thermal conductivity betWeen the
`transport guide and each sample constant over the Whole
`guide; consequently, temperature change of the sample may
`occur during transporting or the samples may differ in
`temperature. Since ?uorescence is detected about the trans
`ported samples one at a time, the same sample must be
`transported repeatedly to monitor change of a ?uorescence
`signal With time for a long time, thus the number of samples
`that can be treated is limited. Further, the risk of contami
`nation (carry-over) betWeen the samples caused by a sample
`splash cannot be excluded.
`The second method described above can solve the prob
`lems of the ?rst method, but may introduce the folloWing
`neW problems:
`First, the conventional apparatus (a) comprises a plurality
`of photosensors, thus the manufacturing costs are increased
`and the space matching the number of photosensors also
`becomes necessary. If an attempt is made to miniaturiZe the
`apparatus, several photosensors can only be installed
`because of the limit of the space; after all, the number of
`samples that can be treated at the same time is only a feW.
`Although use of small-siZed photosensors such as photo
`diodes can also be considered, there is a problem of insuf
`?cient sensitivity to feeble ?uorescence, and it becomes
`necessary to correct the sensitivity of each photodiode.
`Further, the strength of a ?uorescence signal is proportional
`to the excitation light strength and thus if excitation light
`from the light source is divided, detection sensitivity is
`Worsened; this is also a problem.
`Next, the apparatus (b) has insuf?cient sensitivity to
`feeble ?uorescence and thus is not adequate. To augment
`insufficient sensitivity, an element for amplifying the light
`quantity via electron ampli?cation by a microchannel plate
`(so-called image intensi?er) or the like may be used in
`combination, but is used only in special research application
`under the present circumstances because of an extremely
`rise in costs. Since ?uorescence from a Wide range is
`detected as an image, there are also problems of unevenness
`of light quantity detection caused by lens aberration and data
`processing load caused by an enormous data amount.
`With the apparatus (c), the move range is limited because
`of the limit of the bendability of the light guide and
`moreover there is a possibility of breaking light guide. Since
`light communication ef?ciency is changed because the light
`guide is bent, it is dif?cult to make ?uorescence detection
`good in reproducibility. On the other hand, a mechanical
`move of the photosensor also involves a move of attached
`cables, etc., thus the move range is limited and there is a
`possibility of breaking the cable, etc.
`In addition, scanner-type ?uorescence detection apparatus
`as disclosed in Japanese patent Unexamined Publication No.
`2000-088752 (P2000-88752A) and an apparatus as shoWn in
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`3
`FIG. 6 invented as means for solving problems of the
`above-described apparatus are also available. The scanner
`type ?uorescence detection apparatus described in Japanese
`patent Unexamined Publication No. 2000-088752 is as
`folloWs: As shoWn in FIGS. 5AAND 5B, sample vessels are
`arranged like a circular arc and a ring section of a ring-type
`light guide 21 is placed closely facing the sample vessels
`With putting a partition plate 23 therebetWeen and excitation
`light optical means 25 and ?uorescence optical means 26 are
`?xed to the partition plate for rotation integrally, Whereby
`separately gathered ?uorescence signals are communicated
`through the ring-type light guide 21 to a photosensor 22. In
`the scanner-type ?uorescence detection apparatus as shoWn
`in FIG. 6A and 6B, sample vessels are arranged like a
`plurality of circular arcs and a ring-type light guide 31 is
`placed facing the sample vessels With a partition plate 33
`betWeen and excitation light optical means 35 and ?uores
`cence optical means 36 containing at least one light guide
`are ?xed to the partition plate 33 for rotation integrally,
`Whereby separately gathered ?uorescence signals are com
`municated through the ring-type light guide 31 to a photo
`sensor 32.
`The scanner-type ?uorescence detection apparatus as
`shoWn in FIGS. 5A, 5B, 6A and 6B, can solve the conven
`tional problem. HoWever, if a small-siZed and loW-output
`excitation light source is used to furthermore miniaturiZe the
`Whole apparatus, the ?uorescence signal becomes extremely
`feeble and even if a high-sensitivity photosensor such as a
`photomultiplier tube is used, insuf?cient sensitivity may
`result, because the incidence port of the ring-type light guide
`(21, 31) used for communicating the ?uorescence signal
`generally is narroW as several hundred pm and the ?uores
`cence signal communication efficiency is loW. Particularly,
`in the scanner-type ?uorescence detection apparatus as
`shoWn in FIGS. 6A and 6B, the second light guide Which
`rotates is placed in series in addition to the ring-type light
`guide 31 placed still and a signal is communicated
`therebetWeen, thus the scanner-type ?uorescence detection
`apparatus easily falls into insuf?cient sensitivity to
`extremely feeble ?uorescence. If a high-output excitation
`light source such as an argon ion laser is used, the problem
`of insuf?cient sensitivity is resolved, but a large space is
`required for the excitation light source together With a
`control poWer supply, presenting an obstacle to miniaturiZa
`tion of the apparatus.
`Thus, the ?uorescence detection apparatus for monitoring
`a ?uorescence signal in real time, particularly for monitoring
`a ?uorescence signal in real time While incubating a sample
`at a predetermined temperature need satisfy the require
`ments of (a) high-accuracy temperature adjustment, (b)
`rapid treatment of a large number of samples, (c) high
`sensitivity, (d) high reliability (decrease in mechanical
`trouble typi?ed by broken line, moving part operation
`failure, etc., improvement in reproducibility of ?uorescence
`detection, decrease in the risk of carry-over), (e) loW costs
`(simpli?cation of apparatus con?guration, use of no expen
`sive parts in data processing, etc.,),
`miniaturiZation of the
`apparatus, and the like.
`
`SUMMARY OF THE INVENTION
`It is therefore an object of the invention to provide a
`?uorescence detection apparatus satisfying the requirements
`and in particular to a ?uorescence detection apparatus using
`a small-siZed and high-sensitivity optical system of ?uores
`cence analysis, useful for monitoring a large number of ?xed
`samples in real time and a ?uorescence detection apparatus
`provided With an incubation function of temperature control
`means.
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`To the end, according to a ?rst aspect of the invention,
`there is provided a ?uorescence detection apparatus com
`prising a sample holder for ?xing and holding sample
`vessels on a circular arc, a partition plate being joined to
`drive means for rotation on the center of the circular arc, an
`excitation light source, excitation light optical means, and
`?uorescence optical means being ?xed on the partition plate
`for rotation integrally, and a photosensor being mechanically
`discontinued from the drive means and ?xedly placed. In the
`?uorescence detection apparatus,
`(a) the excitation light optical means is placed so as to
`guide excitation light from the excitation light source
`from the rotation center side of the partition plate and
`selectively excite one of the sample vessels,
`(b) the ?uorescence optical means contains a light guide
`for communicating a ?uorescence signal from the
`sample vessel to the photosensor and an incidence end
`of the light guide is placed so as to be able to face the
`sample vessel With the partition plate betWeen so that
`the ?uorescence signal can be gathered and an emission
`end of the light guide is placed so as to be able to face
`the photosensor on the rotation center axis of the
`partition plate, and
`(c) While the excitation light is guided to the sample
`vessels placed on the circular arc in order as the
`partition plate is rotated, ?uorescence is detected
`through the ?uorescence optical means containing the
`light guide at the same time.
`To the end, according to a second aspect of the present
`invention, the ?uorescence detection apparatus as shoWn in
`the ?rst aspect further includes Wavelength dispersion means
`facing the emission end of the light guide for dispersing
`?uorescence into optical paths depending on the Wavelength
`of the ?uorescence signal, Wherein a photosensor is ?xedly
`placed on each of the optical paths, Whereby ?uorescence
`signals having a plurality of Wavelengths can be detected at
`the same time.
`To the end, according to a third aspect of the present
`invention, the ?uorescence detection apparatus as shoWn in
`the ?rst or second aspect further includes a shading plate for
`covering at least the top of one sample vessel, the shading
`plate being ?xed to the partition plate so as to be positioned
`above the ?uorescence incidence end of the light guide
`forming a part of the ?uorescence optical means. Then the
`shading plate is rotated With the partition plate integrally,
`thereby shielding at least the sample vessel under ?uores
`cence measurement in order from extraneous light.
`To the end, according to a fourth aspect of the present
`invention, in the ?uorescence detection apparatus as shoWn
`in the ?rst, second or third aspect, a light emitting diode or
`a semiconductor laser is used as the excitation light source.
`To the end, according to a ?fth aspect of the present
`invention, in the ?uorescence detection apparatus as shoWn
`in the ?rst, second, or third aspect, the light guide being one
`component of the ?uorescence optical means is one optical
`?ber.
`To the end, according to a sixth aspect of the present
`invention, in the ?uorescence detection apparatus as shoWn
`in the second or third aspect, the Wavelength dispersion
`means for dispersing ?uorescence into optical paths depend
`ing on the Wavelength of the ?uorescence signal is a dichroic
`mirror.
`To the end, according to a seventh aspect of the present
`invention, the ?uorescence detection apparatus as shoWn in
`the ?rst, second, or third aspect further includes temperature
`adjustment means for controlling each sample at a prede
`termined temperature.
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`5
`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIGS. 1A and 1B are drawings to show an outline of a
`?uorescence detection apparatus of the invention;
`FIG. 2 is a drawing to show an outline of a ?uorescence
`detection apparatus of the invention that can detect multiple
`wavelengths at the same time;
`FIG. 3 is a drawing to show an outline of a ?uorescence
`detection apparatus of the invention comprising a shading
`plate;
`FIGS. 4A and 4B are drawings to show an outline of a
`?uorescence detection apparatus of the invention compris
`ing temperature adjustment means;
`FIGS. 5A and 5B are a drawing to show an outline of a
`scanner-type ?uorescence detection apparatus in a related
`art;
`FIGS. 6A and 6B is a drawing to show an outline of a
`scanner-type ?uorescence detection apparatus in a related
`art;
`FIGS. 7A and 7B are general views to describe one
`embodiment of a ?uorescence detector of the invention;
`FIG. 8 is a front view to describe in detail a part of the
`?uorescence detection apparatus shown in FIGS. 7A, B;
`FIGS. 9A and 9B are respectively a top view and a front
`view to show the cross section to describe in detail a sample
`holder and temperature adjustment means of the ?uores
`cence detection apparatus shown in FIGS. 7A, B; and
`FIG. 10 is a front view to show the cross section to
`describe in detail a heat insulation case of temperature
`adjustment means, a part of the ?uorescence detection
`apparatus shown in FIGS. 7A, B.
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`DETAILED DESCRIPTION OF THE PREFERED
`EMBODIMENT
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`35
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`A ?uorescence detection apparatus of the invention will
`be discussed in detail with reference to the accompanying
`drawings.
`FIGS. 1A and 1B show an outline of a ?rst embodiment
`of the present invention, a ?uorescence detection apparatus
`using a small-siZed and high-sensitivity optical system of
`?uorescence analysis, useful for monitoring a large number
`of ?xed samples in real time.
`A sample holder (4) comprises hold holes ?tted to the
`outer shape of each sample vessel S on a circular arc for
`?xedly holding the sample vessels (S) each storing a sample
`on the circular arcs. Placement of the sample vessels (S) on
`the circular arcs is not limited to the placement of the sample
`vessels (S) equally spaced from each other as shown in the
`?gure and may be placement of the sample vessels (S)
`unequally spaced from each other. The number of sample
`vessels ?xedly held in the sample holder (4) is not limited
`and may be determined from the length of each circular arc,
`the outer diameter of each sample vessel, etc. Further, the
`shape of the top of the sample holder (4) is not limited to a
`circle and can also be made a polygon such as a quadrangle.
`The sample vessel may be any if it is made of a material
`capable of allowing excitation light and ?uorescence to pass
`through and chemically stable toward the stored sample; it
`can be appropriately selected and used considering the
`sample amount, etc., presented for ?uorescence detection.
`Particularly, to monitor reaction while amplifying a nucleic
`acid as an enZyme in so-called PCR, NASBA, etc., prefer
`ably a sample vessel (S) having a sealing plug is used for the
`purpose of preventing the ampli?ed nucleic acid from scat
`tering.
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`Placed below the ?xedly placed sample holder (4) is a
`partition plate (3) joined to drive means (7) for rotation on
`the center of the circular arc of each sample vessel (S) placed
`on the circular arc. Further, an excitation light source (8),
`excitation light optical means (5), and ?uorescence optical
`means (6) are ?xed to the partition plate (3) and these are
`rotated integrally by the action of the drive means
`Preferably, the partition plate (3) is implemented as a disk
`for stabiliZing rotation. It has a siZe (radius) made larger than
`at least the distance between the circular arc center and the
`sample vessel (S) so as to cut off the ?uorescence optical
`path from the sample vessel to a photosensor (2) except for
`the location where the ?uorescence optical means (6)
`described later exists. However, a circular or slit-like hole is
`made only in the location where the ?uorescence optical
`means (6) described later is ?xed so as to allow a ?uores
`cence signal to pass through. The partition plate (3) can also
`be placed above the sample vessels (S), but a device of
`detaching the partition plate (3) to place the sample vessels
`(S) in the sample holder (4) or the like becomes necessary.
`Further, the sample amount often is small as several ten pl,
`in which case the ?uorescence detection ef?ciency is raised
`if a ?uorescence signal is gathered from the bottom of the
`sample vessel
`For these reasons, preferably the partition
`plate (3) is placed below the sample holder
`The excitation light source (8) may be selected consider
`ing the excitation wavelength of a sample; such an excitation
`light source providing a suf?cient light quantity of excitation
`light arriving at one sample vessel through the excitation
`light optical means (5) is used. To ?x the excitation light
`source (8) on the partition plate (3), preferably the excitation
`light source is small-siZed as much as possible. More
`particularly, a light emitting diode or a semiconductor laser
`can be exempli?ed; a light emitting diode is used in the
`example in FIGS. 1A, B.
`The excitation light optical means (5) ?xed on the parti
`tion plate (3) is means for selecting the wavelength of
`excitation light from light of the excitation light source (8)
`and selectively guiding the excitation light into only one of
`the sample vessels placed like circular arcs. In the apparatus,
`optical means (1) comprising wavelength selection means
`and condensing means is provided. In the example in FIG.
`1B, an interference ?lter is used as the wavelength selection
`means and light can be collected on a speci?c sample vessel
`through an optical lens. The expression “guiding the exci
`tation light into only one of the sample vessels” is not used
`in a strict sense; it is enough to intentionally guide the
`excitation light into one of the sample vessels and, for
`example, if a very small amount of excitation light arrives at
`any other sample vessel by re?ection on the outer wall of the
`one sample vessel, there is no harm.
`The ?uorescence optical means (6) ?xed on the partition
`plate (3) contains at least one light guide (6a); it is means for
`communicating only ?uorescence emitted from the sample
`vessel into which the excitation light is guided as described
`above to the photosensor
`Therefore, a ?uorescence
`incidence end and a ?uorescence emission end of the light
`guide (6a) are placed facing the sample vessel and the
`photosensor (2), respectively. Of course, condensing means
`of an optical lens, etc., intended for improving the ?uores
`cence communication ef?ciency or wavelength selection
`means for selecting ?uorescence wavelength may be
`inserted between the sample vessel (S) and the ?uorescence
`incidence end of the light guide (6a). The condensing means
`or the wavelength selection means may be inserted between
`the ?uorescence signal emission end of the light guide (6a)
`and the photosensor
`In the example in FIG. 1B, the
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`THERMO FISHER EX. 1030
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`

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`US 6,515,743 B1
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`7
`condensing means is inserted betWeen the sample vessel (S)
`and the ?uorescence incidence end of the light guide (6a)
`and the condensing means and the Wavelength selection
`means are inserted betWeen the ?uorescence signal emission
`end of the light guide (6a) and the photosensor
`The ?uorescence signal emission end of the light guide
`(6a) is placed on the rotation center axis of the partition plate
`(3). Thus, if the partition plate (3) joined to the rotation drive
`mechanism (7) is rotated, the position of the ?uorescence
`signal emission end of the light guide (6a) does not change
`and it is made possible to communicate a ?uorescence signal
`to the photosensor (2) With the same efficiency. As the
`optimum light guide, one optical ?ber rich in ?exibility or a
`dense bundle of optical ?bers Whose end faces are aligned
`With a proper metal ?xture at both ends can be used.
`In the apparatus in FIGS. 1A and 1B adopting the
`described con?guration, as the partition plate (3) is rotated,
`excitation light is guided by the excitation light optical
`means (5) into the sample vessels ?xedly held in the sample
`holder (4) one at a time in order. At the same time,
`?uorescence emitted from the sample vessel (S) is detected
`by the photosensor (2) through the ?uorescence optical
`means (6) containing the light guide (6a). Therefore, if the
`detection result of the photosensor (2) is stored While
`rotation of the partition plate (3) is controlled using a
`computer, etc., the intermittent ?uorescence detection result
`of any desired sample held in the sample holder (4) can be
`provided and real-time monitoring can be accomplished.
`Thus, the Whole apparatus can be miniaturiZed by ?xing
`the light source on the partition plate
`Particularly, to use
`an external large-siZed excitation light source such as an
`argon ion laser as in the scanner-type ?uorescence detection
`apparatus as shoWn in FIG. 5A to FIG. 6B, the effect of
`miniaturiZing the apparatus of the invention is very clear
`because a laser body tube and its control poWer supply
`occupy a large volume.
`In the scanner-type ?uorescence detection apparatus as
`shoWn in FIG. 5A to FIG. 6B, the rotation center axis of the
`partition plate (23, 33) overlaps the optical path of excitation
`light and therefore must be avoided as the communication
`optical path of a ?uorescence signal and the ring-type light
`guide (21, 31) comprising optical ?bers arranged like a ring
`at the ?uorescence incidence end is used. Since the ?uores
`cence signal incidence end of the ring-type light guide is
`narroW as several hundred pm, the incidence efficiency of a
`?uorescence signal is loW and the ?uorescence detection
`sensitivity may become insuf?cient. In the invention,
`hoWever, the excitation light source is ?xed on the partition
`plate (3), so that the rotation center axis of the partition plate
`(3) can be used to communicate the ?uorescence signal and
`the ring-type light guide is not required as means for
`communicating the ?uorescence signal and one light guide
`makes it possible to communicate the ?uorescence signal
`With high ef?ciency. Thus, the advantage that high
`sensitivity detection of the ?uorescence signal can be
`accomplished can be provided.
`FIG. 2 shoWs an outline of a second embodiment of the
`present invention, a ?uorescence detection apparatus that
`can detect ?uorescence signals of different Wavelengths at
`the same time, useful for monitoring a large number of ?xed
`samples in real time.
`Wavelength dispersion means (11) is an element for
`dispersing ?uorescence emitted from a light guide (6a) into
`optical paths depending on the Wavelength. In the
`embodiment, a dichroic mirror is used for separating a
`?uorescence signal into re?ected light and transmitted light
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`10
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`15
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`25
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`35
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`45
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`55
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`8
`depending on the Wavelength. The re?ected light is detected
`by a photosensor through an optical means (1) (an interfer
`ence ?lter and a condensing lens) and the transmitted light
`is detected by a photosensor (2) through a total re?ection
`mirror (13), an interference ?lter, and a condensing lens. Of
`course, to detect the transmitted light, the total re?ection
`mirror (13) can also be omitted. Further, for the re?ected
`light or the transmitted light, another dichroic mirror is used
`in combination, Whereby the number of dispersed Wave
`lengths can also be increased. Thus, the ?uorescence signals
`of different Wavelengths can be detected at the same time.
`Use of a diffraction grating is also possible as another
`Wavelength dispersion element (11). Since the diffraction
`grating disperses Wavelength continuously, if as many pho
`tosensors as required are placed in the travel direction of
`light of the target Wavelength, multiple-Wavelength ?uores
`cence detection can be made. It is also made possible to
`measure a ?uorescence spectrum by using CCD or a pho
`todiode array, for example, as the photosensor.
`FIG. 3 shoWs an outline of a third embodiment of the
`present invention, a ?uorescence detection apparatus that
`can prevent disturbance of extraneous light, useful for
`monitoring a large number of ?xed samples in real time.
`To detect a feeble ?uorescence signal, it is important to
`exclude disturbance of extraneous light and a shading plate
`(12) is used for such a purpose. The shading plate (12) may
`be siZed and shaped and have a surface color so that it can
`cover at least the top of one sample vessel (S) and prevent
`extraneous light from entering the vessel. Normally, prefer
`ably the surface color is black. The shading plate (12) is
`?xed to a partition plate (3) so as to be positioned above the
`?uorescence signal incidence end of a light guide (6a)
`forming a part of ?uorescence optical means (6) for rotation
`integrally With rotation of the partition plate
`Thus,
`extraneous light can be prevented from entering at least the
`sample vessel or ?uorescence optical means (6) under
`?uorescence measurement and it is made possible to detect
`a ?uorescence signal stably. Further, it is desirable that the
`shading plate is formed as like beach parasol as shoWn in
`FIG. 3.
`FIGS. 4A and 4B shoW an outline of a fourth embodiment
`of the present invention, a ?uorescence detection apparatus
`provided With an incubation function of temperature control
`means, useful for monitoring a large number of ?xed
`samples in real time. That is, the ?uorescence detection
`apparatus in FIGS. 4A, 4B is provided by adding tempera
`ture adjustment means (9) for controlling each sample at a
`predetermined temperature to the optical system of ?uores
`cence analysis previously described With reference to FIGS.
`1A and 1B.
`The temperature adjustment means (9) for controlling
`each sample at a predetermined temperature uses one heater
`(9a) and one temperature sensor (9b). The top shape of a
`sample holder (4) is made annular, the heater (9a) is put on
`the outer periphery, and the temperature sensor (9b) is built
`in the sample holder (4), Whereby each sample can be
`controlled at a predetermined temperature by thermal con
`duction from the sa