WO2009098867A1 - 蛍光検出装置および蛍光検出方法 - Google Patents
蛍光検出装置および蛍光検出方法 Download PDFInfo
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- WO2009098867A1 WO2009098867A1 PCT/JP2009/000423 JP2009000423W WO2009098867A1 WO 2009098867 A1 WO2009098867 A1 WO 2009098867A1 JP 2009000423 W JP2009000423 W JP 2009000423W WO 2009098867 A1 WO2009098867 A1 WO 2009098867A1
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- lens
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- cell body
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- 238000001514 detection method Methods 0.000 title abstract description 5
- 238000005259 measurement Methods 0.000 claims abstract description 95
- 210000005056 cell body Anatomy 0.000 claims abstract description 57
- 238000012545 processing Methods 0.000 claims abstract description 18
- 238000001917 fluorescence detection Methods 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 23
- 230000003287 optical effect Effects 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 10
- 230000001678 irradiating effect Effects 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 2
- 238000010586 diagram Methods 0.000 description 7
- 239000007850 fluorescent dye Substances 0.000 description 5
- 239000012620 biological material Substances 0.000 description 3
- 239000010453 quartz Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000004163 cytometry Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N21/05—Flow-through cuvettes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N21/0303—Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
- G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
Definitions
- the present invention relates to a fluorescence detection apparatus and a fluorescence detection method for irradiating a measurement object with laser light and measuring fluorescence emitted at that time.
- a flow cytometer used in the medical and biological fields incorporates a fluorescence detection device that receives fluorescence emitted from a fluorescent dye of a measurement object by irradiating laser light and identifies the type of the measurement object.
- a fluorescence detection device that receives fluorescence emitted from a fluorescent dye of a measurement object by irradiating laser light and identifies the type of the measurement object.
- attempts have been made to measure fluorescence using fluorescent dyes in order to examine intracellular local information such as proteins.
- a flow cell (flow cell body) is used as shown in Non-Patent Document 1 below.
- This flow cell is an elongated quartz hollow chamber having a quadrangular cross section, and is a member that transmits laser light and irradiates the cells in the sample with laser light.
- the laser light that has passed through the flow cell is irradiated onto a measurement object that passes through a measurement point in the flow cell, and fluorescence is detected by a separately provided detection system.
- the light intensity distribution of the laser beam is a Gaussian distribution.
- An optical system is constructed in which the beam is condensed and converged into an elliptical shape to increase the intensity and a plurality of cells are not irradiated at a time.
- the laser light passes through two cylindrical condenser lenses before reaching the sample stream through which the cells flow, resulting in an elliptical beam.
- the beam width is adjusted with the first lens, and the beam height is adjusted with the second lens.
- the elliptical beam irradiates cells flowing in an elongated flow cell having a quadrilateral cross section. It is described that when the sample pressure is lowered, the sample flow becomes narrower and passes through a portion where the fluctuation of the light intensity at the center of the laser beam is smaller, so that the measurement resolution is improved.
- Non-Patent Document 1 has a problem that the measurement resolution cannot be improved to the extent that local information in cells such as proteins is examined.
- an object of the present invention is to provide a fluorescence detection apparatus and a fluorescence detection method capable of improving measurement resolution to such an extent that local information in cells such as proteins is examined. .
- the present invention is a fluorescence detection apparatus for irradiating a measurement object with laser light and measuring the fluorescence emitted at that time, wherein the flow cell body is formed with a flow path through which the measurement object flows.
- a laser light source unit that irradiates a laser beam to a measurement object that passes through a measurement point in the flow path; a light receiving unit that receives fluorescence of the measurement object irradiated with the laser beam and outputs a light reception signal; From the light reception signal output from the light receiving unit, a processing unit that outputs an output value of fluorescence intensity,
- a lens is provided so as to cross the optical path of the laser beam, and this lens is a surface perpendicular to the moving direction of the measurement object passing through the measurement point of the measurement object.
- a fluorescence detection device characterized in that, when virtually cut, a cross section of the lens forms a part of a circular shape centered on the measurement point.
- the lens is a spherical lens that forms a part of a spherical shape with the measurement point as a focal position.
- the cross-sectional shape of the flow path formed in the blow cell body is a rectangular shape, and the laser beam is irradiated so that an optical axis is parallel to one side of the rectangular shape, and the rectangular shape,
- the ratio of the length of the other side perpendicular to the one side to the length of the one side parallel to the optical axis of the laser light is preferably 1 to 2.5.
- the length of one side parallel to the optical axis of the laser beam is preferably 30 times or more and 200 times or less with respect to the average diameter of the measurement object.
- the flow cell body and the lens are preferably made of the same material. Further, another lens is provided on the surface of the flow cell body in the fluorescence optical path between the measurement point and the light receiving unit, and the lens cross-section forms a part of a circular shape centered on the measurement point. It is also preferable.
- the measurement object is, for example, a cell
- the laser beam irradiates a part of the cell at the measurement point
- the light receiving unit receives fluorescence emitted from the protein in the cell.
- the present invention is a fluorescence detection method for irradiating a measurement object with laser light and measuring the fluorescence emitted at that time, Flowing a measurement object through a flow path provided in the flow cell body;
- the cross section of the flow cell body when passing through the measurement point of the measurement object and virtually cut along a plane perpendicular to the moving direction of the measurement object, forms a part of a circular shape centering on the measurement point.
- the lens is a spherical lens forming a part of a spherical shape with the measurement point as a focal position.
- a lens is provided on the surface of a flow cell body in which a flow path through which a measurement object flows is formed.
- the cross section of the lens forms a part of a circular shape centered on the measurement point.
- NA number of aperture
- the diameter of the focused beam of laser light can be reduced.
- the diameter of the focused beam of laser light can be efficiently reduced.
- the NA number of the focused beam of laser light
- the NA number of the focused beam of laser light
- the diameter of the focused beam of laser light can be reduced.
- the measurement resolution can be improved to the extent that local information in cells such as proteins is examined.
- (A) And (b) is a figure explaining the flow cell body used for the fluorescence detection apparatus of this invention. It is a figure explaining the state through which a laser beam passes the conventional flow cell body.
- Flow cytometer 12 Sample 20
- Signal processing device 22 Laser light source 22r R light source 22g G light source 22b B light sources 23a 1 , 23a 2 , 26b 1 , 26b 2 dichroic mirror 23c.
- FIG. 1 is a schematic configuration diagram of a flow cytometer 10 using the fluorescence detection device of the present invention.
- the flow cytometer 10 irradiates a sample 12 such as a cell to be measured with laser light, detects fluorescence emitted from a part of the sample 12 such as protein, and performs signal processing (fluorescence detection device) 20.
- an analysis device 80 for analyzing the measurement object in the sample 12 from the processing result obtained by the signal processing device 20.
- the signal processing device 20 irradiates the laser light with a predetermined intensity by irradiating the laser light source unit 22, the light receiving units 24 and 26, the processing unit 28 that outputs the output value of the fluorescence intensity of the sample 12, and the operation of each processing.
- a control unit 29 that performs control management; a pipe line 30 that flows in the sheath liquid that forms a high-speed flow; and a flow cell body 31 that is connected to the end of the pipe line 30 and forms the flow of the sample 12; Have An irradiation point (measurement point) of laser light is formed in the flow cell body 31 in the flow path of the sample 12.
- a recovery container 32 is provided on the outlet side of the flow cell body 31.
- the flow cytometer 10 can also be configured to arrange a cell sorter for separating specific cells or the like in the sample 12 within a short period of time by laser light irradiation and separate them into separate collection containers. .
- a lens system is provided so that the laser beam is focused at a predetermined position in the flow path of the flow cell body 31, and this focusing position is a measurement point of the sample 12.
- FIG. 2 is a diagram illustrating an example of the configuration of the laser light source unit 22.
- the laser light source unit 22 includes an R light source 22r, a G light source 22g, a B light source 22b, dichroic mirrors 23a 1 and 23a 2 , a lens system 23c, laser drivers 34r, 34g and 34b, and a power splitter 35. Configured.
- the R light source 22r, the G light source 22g, and the B light source 22b are laser light emitting portions of visible light of 350 nm to 800 nm.
- the R light source 22r emits mainly the red laser light R with a predetermined intensity.
- the G light source 22g emits green laser light G with a predetermined intensity.
- the B light source 22b emits blue laser light B with a predetermined intensity.
- the dichroic mirrors 23a 1 and 23a 2 transmit laser light in a specific wavelength band and reflect laser light in other wavelength bands.
- the lens system 23 c focuses the laser beam composed of the laser beams R, G, and B on the measurement point in the pipe line 30.
- the laser drivers 34r, 34g, and 34b drive the R light source 22r, the G light source 22g, and the B light source 22b, respectively.
- the power splitter 35 distributes the supplied signal to the laser drivers 34r, 34g, and 34b, respectively.
- a semiconductor laser is used as a light source for emitting these laser beams.
- the dichroic mirror 23a 1 is a mirror that transmits the laser beam R and reflects the laser beam G
- the dichroic mirror 23a 2 is a mirror that transmits the laser beams R and G and reflects the laser beam B.
- the laser drivers 34r, 34g, and 34b are connected to the processing unit 28 and the control unit 29 so as to adjust the intensity of emission of the laser beams R, G, and B.
- the R light source 22r, the G light source 22g, and the B light source 22b oscillate in a predetermined wavelength band so that the laser beams R, G, and B excite the fluorescent dye to emit fluorescence in a specific wavelength band.
- the fluorescent dye excited by the laser beams R, G, and B is attached to the sample 12 such as a biological material to be measured, and when passing through the measurement point of the flow cell body 31 as the measurement object, the laser beam is measured at the measurement point. Fluorescent light is emitted at a specific wavelength when irradiated with R, G, and B.
- the light receiving unit 24 is disposed so as to face the laser light source unit 22 with the flow cell body 31 interposed therebetween, and the sample 12 passes through the measurement point by the forward scattering of the laser light by the sample 12 passing through the measurement point.
- the photoelectric converter which outputs the detection signal of is provided.
- the signal output from the light receiving unit 24 is supplied to the processing unit 28, and is used as a trigger signal informing the timing at which the sample 12 passes the measurement point in the pipe line 30 in the processing unit 28.
- FIG. 3 is a schematic configuration diagram illustrating a schematic configuration of an example of the light receiving unit 26.
- the 3 includes a lens system 26a that focuses a fluorescent signal from the sample 12, dichroic mirrors 26b 1 and 26b 2 , bandpass filters 26c 1 to 26c 3, and a photoelectric converter such as a photomultiplier tube. 27a to 27c.
- the lens system 26a is configured to focus the fluorescence incident on the light receiving unit 26 on the light receiving surfaces of the photoelectric converters 27a to 27c.
- the dichroic mirrors 26b 1 and 26b 2 are mirrors that reflect fluorescence in a wavelength band within a predetermined range and transmit the other fluorescence.
- the reflection wavelength band and the transmission wavelength band of the dichroic mirrors 26b 1 and 26b 2 are set so as to be filtered by the band-pass filters 26c 1 to 26c 3 and to capture fluorescence of a predetermined wavelength band by the photoelectric converters 27a to 27c. .
- the band-pass filters 26c 1 to 26c 3 are filters that are provided in front of the light receiving surfaces of the photoelectric converters 27a to 27c and transmit only fluorescence in a predetermined wavelength band.
- the wavelength band of the transmitted fluorescence is set corresponding to the wavelength band of the fluorescence emitted by the fluorescent dye.
- the photoelectric converters 27a to 27c are sensors provided with photomultiplier tubes, for example, and are sensors that convert light received by the photocathode into electrical signals.
- the control unit 29 is a part that performs laser beam irradiation with a predetermined intensity and performs control management of operation of each process in the processing unit 28.
- the processing unit 28 is a part that performs predetermined signal processing and outputs an output value of fluorescence intensity to the analyzer 80.
- the analyzer 80 uses the output value supplied from the processing unit 28 to specify the type of biological material included in the sample 12 that passes through the measurement point of the flow cell body 31 and the biological material included in the sample 12. It is a device that performs the analysis. In this way, the analyzer 80 obtains, for example, a histogram of the types of biological substances contained in the sample 12 and various characteristics in a short time.
- FIG. 4A is a schematic perspective view of the flow cell body 31, and FIG. 4B is a diagram illustrating a state in which laser light is transmitted through the flow cell body 31.
- the flow cell body 31 is a transparent member having a rectangular parallelepiped shape, and is made of quartz or the like. Laser light is incident from the side surface of the flow cell body 31, passes through the inside of the flow cell body 31, and is focused at the center of the flow path 31 b provided in the flow cell body 30 and extending in the vertical direction from the pipe line 30. This focusing position is the measurement point P.
- This measurement point P is the focal position of the lens system 23 c of the laser light source unit 22.
- a spherical lens 31a having a shape of a part of a sphere having the measurement point P as a focal position is provided on the side surface of the flow cell body 31 on which the laser light is incident. That is, the spherical lens 31a is provided so as to cross the optical path of the laser light.
- the spherical center of the spherical lens 31 a that is, the center of curvature of the spherical lens 31 a coincides with the measurement point P of the sample 12.
- Reference numeral 23c in FIG. 4B denotes a lens system (focusing lens) of the laser light source unit 22 shown in FIG.
- the spherical lens 31a is made of the same material as the flow cell body 31, for example, quartz.
- the spherical lens 31a is provided on the laser light incident surface of the flow cell body 31 in order to reduce the diameter of the focused beam of the laser light and improve the measurement resolution.
- the incident angle of the laser beam incident on the spherical lens 31a is 0 degree.
- NA number of the condensed beam of laser light that passes through the flow cell body 31
- ⁇ is an incident angle of the laser light to the flow cell body 31.
- the laser light that has passed through the lens system 23c is always incident at an incident angle of 0 degrees, so that ⁇ does not change.
- NA becomes the value of the refractive index of the medium ⁇ sin ⁇ .
- NA in the condensed beam passing through the flow cell body 31 is sin ⁇ according to Snell's law.
- the spherical lens 31a when the laser light passes through the flow cell body 31, the spherical lens 31a is provided so that the NA does not decrease at the boundary surface of the flow cell body 31 as in the prior art.
- the spherical lens 31a By providing the spherical lens 31a, it is possible to increase the NA compared to the conventional case according to the following formula. As a result, the diameter of the focused beam can be reduced.
- the following expression is an expression assuming that the laser light has a Gaussian distribution.
- ⁇ is the wavelength of the laser beam
- f is the lens focal length
- D is the aperture diameter of the lens system 23c.
- the spherical lens 31a is provided on one side surface of the rectangular parallelepiped flow cell body 31.
- the flow cell through which the fluorescence passes and reaches the light receiving unit 26 is provided.
- a spherical lens 31 may be provided on another side surface of the body 31. Also in this case, the spherical lens 31 is arranged so that the focal position of the spherical lens 31 comes to the position of the measurement point P.
- the NA is made higher than in the prior art.
- the cross-sectional shape of the flow path 31b formed in the flow cell body 31 is limited as follows. That is, the cross-sectional shape of the flow path 31b provided in the flow cell body 31 is a rectangular shape as shown in FIG. 4B, and the laser beam is irradiated so that the optical axis is parallel to one side of the rectangular shape. Is done. At this time, the ratio of the length L 2 of the other side perpendicular to the one side to the length L 1 of the one side parallel to the optical axis of the laser beam in the rectangular shape of the flow path 31b is 1 to 2.5. It is.
- the position through which the sample 12 passes in the optical axis direction of the laser beam can be regulated. For this reason, the inconvenience of the depth of focus which has become shallower by increasing the NA can be solved by setting the ratio of the rectangular shape of the flow path 31b to 1 to 2.5 and regulating the position where the sample 12 flows. . Further, the length L 1 is preferably 30 to 200 times the average size (diameter) of the sample 12 to be measured.
- the flow cytometer 10 is configured as described above.
- the following fluorescence detection method is performed in which the sample 12 is irradiated with laser light and the fluorescence emitted at that time is measured.
- the sample 12 is caused to flow through the flow path provided in the flow cell body 31 using the sheath liquid.
- the laser beam converged by using the spherical lens 31a provided on the surface of the flow cell body 31 is irradiated to the sample 12 passing through the measurement point in the flow path.
- the spherical lens 31a is a lens whose cross section forms a part of a circular shape centering on the measurement point when it is virtually cut along a plane perpendicular to the moving direction of the sample 12 through the measurement point of the sample 12. .
- the spherical lens 31 forms a part of a spherical shape with the measurement point as the focal position.
- the spherical lens 31a is provided in the flow cell body 31, but the present invention is not limited to this.
- the surface of the flow cell body 31 on which the laser beam is incident passes through the measurement point P of the sample 12 and is virtually cut along a plane perpendicular to the moving direction of the sample 12, the cross section forms a part of a circular shape.
- a lens For example, a cylindrical lens may be used.
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Abstract
Description
細胞内の局所情報を調べるためには、計測分解能を従来に比べて向上させることが必要である。
前記フローセル体の表面には、レーザ光の光路を横切るようにレンズが設けられレンズが設けられ、このレンズは、測定対象物の前記測定点を通る、測定対象物の移動方向と垂直な面で仮想的に切断したとき、前記レンズの断面が前記測定点を中心とした円形形状の一部分を成していることを特徴とする蛍光検出装置を提供する。
また、前記フローセル体と前記レンズは同じ材料で構成されていることが好ましい。
さらに、前記測定点と前記受光部との間の蛍光の光路中の前記フローセル体の表面に、レンズの断面が前記測定点を中心とした円形形状の一部分を成した別のレンズが設けられていることも好ましい。
測定対象物を、フローセル体に設けられた流路に流すステップと、
測定対象物の測定点を通り、測定対象物の移動方向と垂直な面で仮想的に切断したとき、断面が前記測定点を中心とした円形形状の一部分を成している、前記フローセル体の表面に設けられたレンズを用いて収束させたレーザ光を、前記流路中の測定点を通過する測定対象物に対して照射させるステップと、
レーザ光の照射された測定対象物の蛍光を受光して受光信号を出力するステップと、
出力した前記受光信号から、蛍光強度の出力値を出力するステップと、を有することを特徴とする蛍光検出方法を提供する。
また、上記レンズを用いて蛍光を検出する方法においても、従来に比べてNA(開口数)を高めることができ、レーザ光の集光ビームの直径を小さくすることができる。特に、測定点を焦点位置とする球体形状の一部分を成す球面レンズを用いることで、レーザ光の集光ビームの直径を効率よく小さくすることができる。
したがって、タンパク質等の細胞内の局所情報を調べる程度に測定分解能を向上することができる。
12 試料
20 信号処理装置
22 レーザ光源部
22r R光源
22g G光源
22b B光源
23a1,23a2,26b1,26b2 ダイクロイックミラー
23c.26a レンズ系
24,26 受光部
26c1,26c2,26c3 バンドパスフィルタ
27a~27c 光電変換器
28 処理部
29 制御部
30 管路
31 フローセル体
31a 球面レンズ
31b 流路
32 回収容器
34r,34g,34b レーザドライバ
80 分析装置
図1は、本発明の蛍光検出装置を用いたフローサイトメータ10の概略構成図である。
フローサイトメータ10は、レーザ光を測定対象とする細胞等の試料12に照射し、試料12中のタンパク質等の一部分から発する蛍光を検出して信号処理する信号処理装置(蛍光検出装置)20と、信号処理装置20で得られた処理結果をから試料12中の測定対象物の分析を行なう分析装置80とを有する。
レーザ光源部22は、R光源22r、G光源22g、B光源22bと、ダイクロイックミラー23a1、23a2と、レンズ系23cと、レーザドライバ34r,34gおよび34bと、パワースプリッタ35と、を有して構成される。
R光源22r、G光源22g、B光源22bは、350nm~800nmの可視光の、レーザ光を出射する部分で、R光源22rは、主に赤色のレーザ光Rを所定の強度で出射する。G光源22gは、緑色のレーザ光Gを所定の強度で出射する。B光源22bは、青色のレーザ光Bを所定の強度で出射する。
ダイクロイックミラー23a1、23a2は、特定の波長帯域のレーザ光を透過し、他の波長帯域のレーザ光を反射する。
レンズ系23cは、レーザ光R,GおよびBからなるレーザ光を管路30中の測定点に集束させる。レーザドライバ34r,34gおよび34bは、R光源22r、G光源22gおよびB光源22bのぞれぞれを駆動する。
パワースプリッタ35は、供給された信号をレーザドライバ34r,34gおよび34bにそれぞれ分配する。
これらのレーザ光を出射する光源として例えば半導体レーザが用いられる。
この構成によりレーザ光R,GおよびBが合成されて、測定点を通過する試料12を照射する照射光となる。
図3は、受光部26の一例の概略の構成を示す概略構成図である。
レンズ系26aは、受光部26に入射した蛍光を光電変換器27a~27cの受光面に集束させるように構成されている。
ダイクロイックミラー26b1,26b2は、所定の範囲の波長帯域の蛍光を反射させて、それ以外は透過させるミラーである。バンドパスフィルタ26c1~26c3でフィルタリングして光電変換器27a~27cで所定の波長帯域の蛍光を取り込むように、ダイクロイックミラー26b1,26b2の反射波長帯域および透過波長帯域が設定されている。
処理部28は、所定の信号処理を行って蛍光強度の出力値を分析装置80に出力する部分である。
フローセル体31は、長方体を成した透明部材であり、石英等によって作られている。フローセル体31の側面からレーザ光が入射され、フローセル体31の内部を通過し、フローセル体30に設けられた、管路30から縦方向に延びる流路31bの中心で集束するようになっており、この集束位置が測定点Pとなっている。この測定点Pは、レーザ光源部22のレンズ系23cの焦点位置である。
このように、フローセル体31のレーザ光の入射面に球面レンズ31aを設けるのは、レーザ光の集光ビームの直径を小さくし、測定分解能を向上させるためである。
具体的には、フローセル体31を通過するレーザ光の集光ビームのNAは、(媒質の屈折率)×sinθで表される。ここで、θはレーザ光のフローセル体31への入射角度である。このとき、球面レンズ31aでは、レンズ系23cを通過したレーザ光は常に入射角度0度で入射するのでθは変化せず、球面レンズ31aを通過したときNAは媒質の屈折率×sinθの値になる。一方、図5に示す従来例のように、球面レンズ31aがない場合、レーザ光がフローセル体31の側面に入射する入射角度は、レーザ光の光軸の部分を除いて、0度ではない。このため、フローセル体31を通過する集光ビームにおけるNAはスネルの法則よりsinθとなる。以上より、本発明では、フローセル体31をレーザ光が通過するとき、フローセル体31の境界面でNAが従来のように低下しないように、球面レンズ31aを設ける。
球面レンズ31aを設けることにより、下記式に従って、従来に比べてのNAを増大させることができる。これによって集光ビームの直径を小さくすることができる。下記式は、レーザ光がガウシアン分布を想定した式である。
ここで、λはレーザ光の波長、fはレンズ焦点距離、Dはレンズ系23cの開口径である。
なお、本実施形態の球面レンズ31aは、直方体形状をしたフローセル体31の1つの側面に設けるが、受光部26の蛍光の測定分解能を向上させるために、蛍光が通過し受光部26に至るフローセル体31の別の側面に球面レンズ31を設けてもよい。この場合においても、測定点Pの位置に球面レンズ31の焦点位置が来るように配置する。
焦点深度z=0.64/(NA)2
すなわち、フローセル体31に設けられた流路31bの断面形状は、図4(b)に示すように矩形形状であり、レーザ光は、光軸がこの矩形形状の一辺に平行になるように照射される。このとき、流路31bの矩形形状の、レーザ光の光軸に平行な一辺の長さL1に対する、この一辺に対して直交する他辺の長さL2の比率が、1~2.5である。このような比率の矩形形状の流路31bを形成することで、レーザ光の光軸方向において試料12が通過する位置を規制することができる。このため、NAを高くすることで浅くなった焦点深度の不都合を、流路31bの矩形形状の比率を1~2.5とし、試料12の流れる位置を規制することにより、解決することができる。また、測定する試料12の平均サイズ(直径)に対して長さL1を30~200倍とすることが好ましい。
フローサイトメータ10は以上のように構成される。
試料12を、フローセル体31に設けられた流路にシース液を用いて流す。そして、フローセル体31の表面に設けられた球面レンズ31aを用いて収束させたレーザ光を、流路中の測定点を通過する試料12に対して照射させる。球面レンズ31aは、試料12の測定点を通り、試料12の移動方向と垂直な面で仮想的に切断したとき、断面が測定点を中心とした円形形状の一部分を成しているレンズである。このとき、レーザ光の照射された試料12の発する蛍光を受光して受光信号を出力する。出力した受光信号から、蛍光強度の出力値を出力する。
なお、球面レンズ31は、測定点を焦点位置とする球体形状の一部分を成している。
Claims (9)
- 測定対象物にレーザ光を照射し、そのとき発する蛍光を測定する蛍光検出装置であって、
測定対象物が流れる流路が形成されたフローセル体と、
流路中の測定点を通過する測定対象物に対してレーザ光を照射するレーザ光源部と、
レーザ光の照射された測定対象物の蛍光を受光して受光信号を出力する受光部と、
前記受光部から出力した受光信号から、蛍光強度の出力値を出力する処理部と、を有し、
前記フローセル体の表面には、レーザ光の光路を横切るようにレンズが設けられ、このレンズは、測定対象物の測定点を通る、測定対象物の移動方向と垂直な面で仮想的に切断したとき、前記レンズの断面が前記測定点を中心とした円形形状の一部分を成していることを特徴とする蛍光検出装置。 - 前記レンズは、前記測定点を焦点位置とする球体形状の一部分を成す球面レンズである請求項1に記載の蛍光検出装置。
- 前記ブローセル体に形成される前記流路の断面形状は、矩形形状であり、前記レーザ光は、光軸がこの矩形形状の一辺に平行になるように照射され、
前記矩形形状の、前記レーザ光の光軸に平行な一辺の長さに対する、この一辺に対して直交する他辺の長さの比率が、1~2.5である請求項1または2に記載の蛍光検出装置。 - 前記レーザ光の光軸に平行な一辺の長さは、測定対象物の平均直径に対して30倍以上200倍以下の長さである請求項3に記載の蛍光検出装置。
- 前記フローセル体と前記レンズは同じ材料で構成されている請求項1~4のいずれか1項に記載の蛍光検出装置。
- 前記測定点と前記受光部との間の蛍光の光路中の前記フローセル体の表面に、レンズの断面が前記測定点を中心とした円形形状の一部分を成した別のレンズが設けられている請求項1~5のいずれか1項に記載の蛍光検出装置。
- 測定対象物は細胞であり、
前記レーザ光は、前記測定点において細胞内の一部分を照射し、
前記受光部は、細胞内のタンパク質が発する蛍光を受光する請求項1~6のいずれか1項に記載の蛍光検出装置。 - 測定対象物にレーザ光を照射し、そのとき発する蛍光を測定する蛍光検出方法であって、
測定対象物を、フローセル体に設けられた流路に流すステップと、
測定対象物の測定点を通り、測定対象物の移動方向と垂直な面で仮想的に切断したとき、断面が前記測定点を中心とした円形形状の一部分を成している、前記フローセル体の表面に設けられたレンズを用いて収束させたレーザ光を、前記流路中の測定点を通過する測定対象物に対して照射させるステップと、
レーザ光の照射された測定対象物の蛍光を受光して受光信号を出力するステップと、
出力した前記受光信号から、蛍光強度の出力値を出力するステップと、を有することを特徴とする蛍光検出方法。 - 前記レンズは、前記測定点を焦点位置とする球体形状の一部分を成す球面レンズである請求項8に記載の蛍光検出方法。
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