WO2009098868A1 - 蛍光検出装置及び蛍光検出方法 - Google Patents
蛍光検出装置及び蛍光検出方法 Download PDFInfo
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- WO2009098868A1 WO2009098868A1 PCT/JP2009/000424 JP2009000424W WO2009098868A1 WO 2009098868 A1 WO2009098868 A1 WO 2009098868A1 JP 2009000424 W JP2009000424 W JP 2009000424W WO 2009098868 A1 WO2009098868 A1 WO 2009098868A1
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- light
- fluorescence
- signal
- measurement object
- laser
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- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000005259 measurement Methods 0.000 claims abstract description 82
- 238000012937 correction Methods 0.000 claims abstract description 43
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 238000012545 processing Methods 0.000 claims description 33
- 230000003287 optical effect Effects 0.000 claims description 10
- 230000001678 irradiating effect Effects 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 210000005056 cell body Anatomy 0.000 description 10
- 238000010586 diagram Methods 0.000 description 9
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000012620 biological material Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000004065 semiconductor 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
- G01N21/62—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
-
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
-
- 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
- G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N2021/4704—Angular selective
- G01N2021/4707—Forward scatter; Low angle scatter
-
- 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
- G01N2021/6482—Sample cells, cuvettes
Definitions
- the present invention relates to a fluorescence detection apparatus and a fluorescence detection method for irradiating a measurement object flowing in a flow path 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 from a fluorescent dye of a measurement object by irradiating laser light and identifies the type of the measurement object. .
- the light intensity of the laser light used in the fluorescence detection device generally has a Gaussian distribution in which the center is high and the intensity decreases as it goes outward.
- the measurement object is irradiated with a central portion where the light intensity of the laser light having a Gaussian distribution is substantially constant.
- the portion of the Gaussian distribution having a constant light intensity is expanded by expanding the laser beam.
- the following non-patent document 1 describes a flow cytometer.
- this flow cytometer in order to obtain high measurement resolution, the light intensity of the laser beam is stable, the sheath liquid that is made to flow by including the measurement object is adjusted to reduce the diameter of the flow, and It is pointed out that the flow must be laminar.
- the light intensity of the laser light described above must be stable, the diameter of the sheath liquid flow should be reduced, and the flow should be layered. Flowing alone is not enough.
- the laser light beam can be expanded to expand a portion having a certain intensity used for measurement of the measurement object, but the outer portion of the laser light is not used for irradiation of the measurement object. Use efficiency of laser light is poor.
- the present invention improves the use efficiency of laser light in a fluorescence detection apparatus that irradiates a measurement object flowing in a flow path with laser light and measures the fluorescence emitted at that time.
- An object of the present invention is to provide a fluorescence detection apparatus and a fluorescence detection method capable of obtaining a high resolution result using the obtained measurement data.
- the present invention is a fluorescence detection device for irradiating a measurement object flowing in a flow path with a laser beam and measuring the fluorescence emitted at that time.
- the optical system for condensing the forward scattered light of the laser light from the measurement object, and the timing at which the measurement object passes the measurement point by receiving the collected forward scattered light And a plurality of parallel arrangements in the direction of the optical axis of the laser beam and the direction of the flow of the measurement object in the flow path in order to detect the focusing position of the collected forward scattered light
- a first light-receiving unit, a second light-receiving unit that receives fluorescence of the measurement object irradiated with laser light through a condenser lens and outputs a light-receiving signal, and the first light-receiving unit Detection signal output from the trigger signal To the second on the basis of the light reception signal outputted to the detection signal and the light receiving unit, to start the data processing includes a processing unit that outputs an
- the processing unit uses the correction table that associates the focus position and the correction coefficient for correcting the received light signal using information on the light intensity distribution of the laser light irradiated to the measurement object, and calculates the correction coefficient. It is preferable to obtain.
- a shielding plate for shielding the laser light in the vicinity of the optical axis may be provided between the flow path and the first light receiving unit so that direct light of the laser light is not irradiated onto the first light receiving unit.
- the present invention is a fluorescence detection method for irradiating a measurement object flowing in a flow path with laser light and measuring the fluorescence emitted at that time, so that the measurement object passes through a measurement point in the flow path.
- Flowing the measurement object and irradiating the measurement object with laser light, and detecting a focusing position where forward scattered light of the laser light is condensed through the optical system when the measurement object passes the measurement point A step of generating a detection signal; a step of receiving the fluorescence of the measurement object irradiated with the laser light through a condenser lens and outputting a light reception signal; and a focusing position of the forward scattered light from the detection signal, Obtaining a correction coefficient for correcting the received light signal from the focusing position, and calculating the fluorescence intensity by correcting the received light signal using the correction coefficient.
- a correction table in which a focusing position and a correction coefficient for correcting the light reception signal are associated with each other using information on the light intensity distribution of the laser light irradiated on the measurement object is used.
- a plurality of measurement objects are sequentially flowed in the flow path, and a frequency distribution of fluorescence intensity is created using the corrected light reception signal. Furthermore, it is also preferable to have a step of obtaining the size of the measurement object using the intensity of the scattered light signal of the forward scattered light and the focusing position of the forward scattered light.
- the first light-receiving unit that notifies the timing at which the measurement object passes the measurement point by collecting the forward scattered light of the laser beam from the measurement object.
- a plurality of detectors arranged in parallel in the direction of the optical axis of the laser light and the direction perpendicular to the direction of the flow of the measurement object in the flow path are provided.
- the processing unit specifies the condensing position of the forward scattered light from the detection signal output from the first light receiving unit, and corrects the light receiving signal output from the second light receiving unit from this converging position. And a received light signal can be corrected using the correction coefficient.
- the peripheral part where the light intensity sharply decreases outside is used for irradiation of the measurement object. Therefore, the use efficiency of the laser beam can be increased.
- a sharp frequency distribution small dispersion
- a high-resolution result can be obtained to such an extent that the two peaks can be distinguished.
- flow cytometer 12 sample 20 signal processor 22 laser light source unit 22r R light source 22 g G light 22b B light source 23a 1, 23a 2, 26b 1 , 26b 2 dichroic mirrors 23c lens system 24, 26 light-receiving portion 24a condenser lens 24b forward Scattering detection unit 24c Shielding plate 24d Detector 26a Focusing lenses 26c 1 , 26c 2 , 26c 3 band pass filters 27a to 27c Photoelectric converter 28 Processing unit 29 Control unit 30 Pipe line 31 Flow cell body 32 Recovery containers 34r, 34g, 34b Laser Driver 35 Power splitter 80 Analyzer
- 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 and performs signal processing, and a signal processing device 20.
- an analysis device 80 for analyzing the measurement object in the sample 12 from the processing result obtained by the processing device 20.
- the signal processing device 20 includes a laser light source unit 22, light receiving units 24 and 26, a processing unit 28, a control unit 29, a conduit 30, and a flow cell body 31.
- the processing unit 28 outputs the output value of the fluorescence intensity of the sample 12.
- the control unit 29 irradiates the laser beam with a predetermined intensity, and controls and manages the operation of each process.
- the pipe line 30 flows the sample 12 in a sheath liquid that forms a high-speed flow.
- the flow cell body 31 is connected to the end of the pipe line 30 to form a flow of the sample 12, and a laser beam measurement point is created in the flow path.
- 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 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 pipe 30 and the flow cell body 31 interposed therebetween, and the sample 12 is measured at the measurement point by the forward scattering of the laser light by the sample 12 passing through the measurement point. And a photoelectric converter that outputs a detection signal indicating that the light passes through.
- the signal output from the light receiving unit 24 is supplied to the processing unit 28 and the control unit 29, and is used as a trigger signal that notifies the timing at which the sample 12 passes through the measurement point in the pipe 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 24.
- the light receiving unit 24 includes a condensing lens 24a that condenses laser light that strikes the sample 12 and scatters forward, a multi-channel forward scattered light detection unit 24b that detects the condensed light, and a shielding plate 24c.
- the condensing lens 24a is used to condense the laser light that is irradiated to the sample 12 and scattered forward (forward in the direction of travel of the laser light, right side in FIG. 3).
- a plurality of detectors 24d are arranged in parallel in a direction perpendicular to the irradiation direction of the laser light and perpendicular to the direction in which the sample 12 flows (perpendicular to the paper surface in FIG. 3).
- Each of the detectors 24 d is connected so that a detection signal is output to the processing unit 28 and the control unit 29.
- photodiodes having the same configuration are used.
- a plurality of detectors 24d are arranged in parallel to obtain information on positional deviation when the sample 12 passes with a positional deviation from the center of a preset measurement point (laser beam focusing point). Because. Specifically, when the sample 12 is displaced in the X direction in FIG. 3, the forward scattered light of the laser light is positioned below the center position A of the scattered light detection unit 24b (the arrow in FIG. 3). Focusing at a position in the direction of. When the sample 12 is displaced in the direction opposite to the X direction in FIG. 3, the forward scattered light of the laser light is focused at a position on the upper side in the figure from the center position A of the scattered light detection unit 24b.
- This focusing position changes according to the amount of positional deviation of the sample 12 in the X direction. Therefore, the focusing position can be known depending on which detector 24d detects the maximum intensity of the measured scattered light. From this, it can be known how much the sample 12 to be measured has shifted with respect to the measurement point.
- the detected detection signal of the detector 24d is supplied to the processing unit 28 and the control unit 29. Since the detection signal is output from each detector 24d, it is possible to know which detector 24d has detected the maximum intensity of the scattered light by specifying the detection signal indicating the maximum value.
- the detection signal is used as a trigger signal for starting data processing.
- the shielding plate 24c is used to shield the direct light of the irradiated laser light so that the detector 24d does not receive the direct light.
- the light receiving unit 26 is in a direction perpendicular to the flow of the sample 12 on a plane perpendicular to the optical axis of the laser light emitted from the laser light source unit 22, that is, a direction perpendicular to the irradiation direction of the laser light.
- a photoelectric converter that is arranged in a direction perpendicular to the moving direction of the sample 12 in the flow path of the flow cell body 31 and receives fluorescence emitted from the sample 12 irradiated at the measurement point.
- the light receiving unit 26 is arranged in a direction perpendicular to the emitting direction of the laser light emitted from the laser light source unit 22 and perpendicular to the moving direction of the sample 12 in the flow path of the flow cell body 31. And a photoelectric converter that receives fluorescence emitted from the sample 12 irradiated at the measurement point.
- FIG. 4 is a schematic configuration diagram illustrating a schematic configuration of an example of the light receiving unit 26.
- the lens system 26a includes a lens system 26a that focuses a fluorescent signal from the sample 12, dichroic mirrors 26b 1 and 26b 2 , band-pass 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 that include a sensor including, for example, a photomultiplier tube, and convert light received by the photoelectric surface into an electrical signal.
- the control unit 29 is a part that performs laser beam irradiation with a predetermined intensity and controls and manages the operation of each process in the processing unit 28 based on the trigger signal supplied from the light receiving unit 24.
- 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 processing unit 28 starts predetermined data processing based on the detection signal supplied from the light receiving unit 24.
- the processing unit 28 specifies from which detector 24d the detection signal supplied from the light receiving unit 24 takes the maximum value, thereby specifying the focusing position of the forward scattered light, From this specific position, a correction coefficient for correcting the light reception signal output from the light receiving unit 26 is obtained, and the light reception signal is corrected using this correction coefficient.
- the processing unit 28 performs correction using a correction table stored in advance so as to collectively determine the intensity of the laser light from the focusing position of the detector 24d and to obtain a correction coefficient corresponding to the intensity.
- This correction table is set by associating the focusing position and the correction coefficient for correcting the received light signal using the information on the light intensity distribution of the laser beam irradiated onto the sample 12.
- FIGS. 5A to 5E are diagrams for explaining the necessity of correcting the received light signal.
- the received light signal is calculated by the analyzer 80, which will be described later, by calculating the frequency distribution of the sample 12 with respect to the fluorescence intensity as shown in FIG. 5D, and whether or not specific fluorescence has been measured from this frequency distribution. Used to determine At this time, when two types of fluorescence exist, two peaks are formed in the frequency distribution. When the fluorescence intensities of these two peaks are close to each other, it is necessary that the peaks have a narrow width (small dispersion) in order to be able to distinguish the two peaks. As shown in FIG. 5E, when the peak width of one peak is wide (variance is large), the two peaks cannot be distinguished.
- FIG. 5E shows a frequency distribution when the above correction is not performed.
- a correction coefficient having a reciprocal distribution of the light intensity distribution of the laser beam at this position is used according to the positions A to C through which the sample 12 passes, and this correction coefficient is used as a light reception signal. Multiply by.
- the position of the sample 12 varies with a distribution as shown in FIG. 5C
- the received light signal is corrected using the correction coefficient as described above, and therefore the frequency distribution when no correction is made is shown in FIG.
- the peak width becomes narrower as shown in FIG.
- correction simply multiplies the received light signal by a correction coefficient. This is because a portion in which the fluorescence intensity changes linearly according to the light intensity of the laser beam irradiated is preferably used. However, the present invention is not limited to this. At least the intensity of the fluorescence has a corresponding relationship with the light intensity of the irradiated laser beam, and the correction coefficient may be determined using this relationship.
- the analyzer 80 creates a frequency distribution as shown in FIG. 5D using the corrected received light signal supplied from the processing unit 28 and is included in the sample 12 passing through the measurement point of the flow cell body 31.
- This is an apparatus that identifies the type of biological material and the like and analyzes the biological material contained in the sample 12.
- the forward scattered light emitted from the sample 12 is detected by the detector 24d of the scattered light detection unit 24b. Since the convergence position of the forward scattered light is known from the detection signal generated by the detector 24d, a correction coefficient is obtained using a correction table held by the processing unit 28 based on the convergence position. By multiplying the light reception signal of the light receiving unit 26 by this correction coefficient, the light reception signal is corrected. The corrected light reception signal is supplied to the analyzer 80, and a frequency distribution as shown in FIG. 5D is created.
- the processing unit 28 corrects the received light signal using the correction table using the light intensity distribution of the laser light, so that the analysis device 80 can obtain a frequency distribution with a narrow peak width. .
- the processing unit 28 corrects the received light signal using the correction table using the light intensity distribution of the laser light, so that the analysis device 80 can obtain a frequency distribution with a narrow peak width.
- correction is made using this distribution, so that it is not necessary to use only the central part of the laser light having a constant light intensity for fluorescence measurement as in the past.
- the outer portion of the central portion of the laser beam that has not been used for the conventional measurement can also be used for the fluorescence measurement. For this reason, a laser beam can be used efficiently. Further, the position through which the sample 12 flows can be examined.
- the light intensity of the laser beam at the position of the sample 12 is known from the detection signal of the detector 24d.
- the light intensity of the forward scattered light is obtained as a result of multiplying the light intensity of the laser light at the position of the sample 12 and the size of the sample 12, the sample measured using the light intensity of the known laser light. It is also possible to know 12 approximate sizes.
- the analyzer 80 may perform the process of calculating the approximate size of the measured sample 12 as described above. At this time, the size of the sample 12 is obtained using the intensity of the scattered light signal of the forward scattered light generated by the light receiving unit 24 and the focal position of the forward scattered light.
- the focus position of the forward scattered light is known, the position shift of the measurement point through which the sample 12 passes is obtained from this focus position, and the light intensity of the laser beam irradiated on the position-shifted sample 12 is calculated. I can know.
- the size of the sample 12 can be obtained from the light intensity of the laser light and the intensity of the scattered light signal of the forward scattered light.
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Abstract
Description
例えば、レーザ光の光束を拡げて、測定対象物の測定に用いる一定の強度を備える部分を拡げることはできても、レーザ光の外側の部分は、測定対象物の照射に用いていないため、レーザ光の使用効率が悪い。
前記処理部は、前記第1の受光部から出力された検知信号から前方散乱光の集束位置を特定し、この集束位置から、前記第2の受光部から出力された受光信号を補正するための補正係数を求め、この補正係数を用いて前記受光信号を補正することを特徴とする蛍光検出装置を提供する。
さらに、補正された受光信号を用いて、蛍光強度の頻度分布を作成する分析部を備えることが好ましい。
さらに、前記第1の受光部にて生成される前記前方散乱光の散乱光信号の強度と前記前方散乱光の集束位置とを用いて測定対象物のサイズを求める分析部を備えることも好ましい。
さらに、前記前方散乱光の散乱光信号の強度と前記前方散乱光の集束位置とを用いて測定対象物のサイズを求めるステップを有することも好ましい。
また、受光信号を補正するので、分析部で作成する蛍光強度に対する頻度分布において、シャープな(分散が小さい)頻度分布を得ることができる。このため、頻度分布において、2つのピークが近接して存在する場合でも、2つのピークを判別できる程度に、高分解能の結果を得ることができる。
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 レンズ系
24,26 受光部
24a 集光レンズ
24b 前方散乱検出ユニット
24c 遮蔽板
24d 検出器
26a 集束レンズ
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
32 回収容器
34r,34g,34b レーザドライバ
35 パワースプリッタ
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中の一部分から発する蛍光を検出して信号処理をする信号処理装置(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。
処理部28は、試料12の蛍光強度の出力値を出力する。制御部29は、所定の強度でレーザ光を照射させ、各処理の動作の制御管理を行う。管路30は、高速流を形成するシース液に含ませて試料12を流す。フローセル体31は、管路30の端に接続され、試料12のフローを形成し、このフローの経路にレーザ光の測定点をつくる。
フローセル体31の出口側には、回収容器32が設けられている。フローサイトメータ10には、レーザ光の照射により短時間内に試料12中の特定の細胞等を分離するためのセル・ソータを配置して別々の回収容器に分離するように構成することもできる。
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。
受光部24は、試料12に当たって前方散乱するレーザ光を集光する集光レンズ24aと、集光した光を検出する多チャンネルの前方散乱光検出ユニット24bと、遮蔽板24cと、を有する。
前方散乱光検出ユニット24bは、レーザ光の照射方向と直交し、かつ、試料12の流れる方向(図3中紙面に垂直方向)と直交する方向に、複数の検出器24dが並列配置されている。検出器24dのそれぞれは、検出信号が処理部28及び制御部29に出力されるように接続されている。検出器24dは、例えば、いずれも同じ構成をしたフォトダイオードが用いられる。このように、検出器24dを複数並列配置したのは、試料12が予め設定された測定点(レーザ光の集束点)の中心に対して位置ずれして通過するときの位置ずれの情報を得るためである。具体的には、試料12が、図3中、X方向に位置ずれした場合、レーザ光の前方散乱光は、散乱光検出ユニット24bの中心位置Aより図中下側の位置(図3中矢印の方向の位置)で集束する。試料12が、図3中、X方向と反対側方向に位置ずれした場合、レーザ光の前方散乱光は、散乱光検出ユニット24bの中心位置Aより図中上側の位置で集束する。この集束位置は、試料12のX方向の位置ずれ量に応じて変化する。したがって、計測した散乱光の最大強度がどの検出器24dで検出されたかによって、集束位置を知ることができる。これより、測定対象の試料12が測定点に対してどのくらい位置ずれして流れたかを知ることができる。測定した検出器24dの検知信号が処理部28及び制御部29に供給される。検知信号は、検出器24dそれぞれから出力されるので、最大値を示す検知信号を特定することで、散乱光の最大強度がどの検出器24dで検出されたか、知ることができる。また、検知信号は、データ処理の開始のためにトリガー信号として用いられる。
遮蔽板24cは、照射されたレーザ光の直接光が検出器24dで受光されないように、この直接光を遮蔽するために用いられる。
図4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。
処理部28では、受光部24から供給された検知信号に基づいて所定のデータ処理を開始する。処理部28は、受光部24から供給された検出信号のうち、最大の値を取るものが、どの検出器24dからのものであるか特定し、これによって前方散乱光の集束位置を特定し、この特定位置から、受光部26から出力された受光信号に補正をするための補正係数を求め、この補正係数を用いて受光信号を補正する。
処理部28は、検出器24dの集束位置からレーザ光の強度を求め、この強度に応じた補正係数を求める処理を一括して行うために、予め記憶されている補正テーブルを用いて補正を行う。この補正テーブルは、試料12に照射されるレーザ光の光強度分布の情報を用いて集束位置と受光信号を補正するための補正係数とを関係付けて設定されたものである。
本発明では、受光信号は、後述する分析装置80において、図5(d)に示すような蛍光強度に対する試料12の頻度分布を算出し、この頻度分布から、特定の蛍光が測定されたか否かを判別するために使用する。このとき、蛍光が2種類存在する場合、頻度分布において2つのピークを形成する。この2つのピークの蛍光強度が近接しているとき、この2つのピークが判別できるためには、ピークの幅が狭いこと(分散が小さいこと)が必要である。図5(e)のように、1つのピークのピーク幅が広い(分散が大きい)場合、2つのピークは判別できない。図5(e)は、上述の補正をしない場合の頻度分布である。
補正は単純に補正係数を受光信号に乗算するものである。これは、蛍光の強度が照射されるレーザ光の光強度に応じて線形的に変化する部分を好適に用いるからである。しかし、本発明ではこれに限定されない。少なくとも蛍光の強度が照射されるレーザ光の光強度と対応関係にあり、この関係を用いて補正係数を定めるとよい。
補正された受光信号は、分析装置80に供給されて、図5(d)に示すような頻度分布が作成される。
又、試料12の流れる位置を調べることもできる。検出器24dの検出信号から試料12の位置におけるレーザ光の光強度が既知となっている。一方、前方散乱光の光強度は、試料12の位置におけるレーザ光の光強度と試料12のサイズの乗算した結果として得られるものなので、既知のレーザ光の光強度を用いて、測定された試料12の概略のサイズを知ることもできる。このような測定された試料12の概略のサイズの算出処理を、分析装置80は行ってもよい。このとき、受光部24にて生成される前方散乱光の散乱光信号の強度と、前方散乱光の集束位置とを用いて試料12のサイズを求める。具体的には、前方散乱光の集束位置は既知となるので、この集束位置から試料12が通過する測定点の位置ずれを求め、この位置ずれした試料12の照射されるレーザ光の光強度を知ることができる。このレーザ光の光強度と、前方散乱光の散乱光信号の強度とから試料12のサイズを求めることができる。
Claims (9)
- 流路中を流れる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、
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、
測定対象物からのレーザ光の前方散乱光を集光する光学系と、集光した前記前方散乱光を受光することによって、測定対象物が測定点を通過するタイミングを知らせるとともに、集光した前方散乱光の集束位置を検知するために、前記レーザ光の光軸の方向及び前記流路中の測定対象物の流れの方向と直交する方向に並列配置された複数の検出器と、を備える第1の受光部と、
レーザ光の照射された測定対象物の蛍光を集光レンズを通して受光して受光信号を出力する第2の受光部と、
前記第1の受光部から出力した検知信号をトリガー信号として、前記第2の受光部から出力した前記受光信号と前記検知信号とに基づいて、データ処理を開始し、蛍光強度の出力値を出力する処理部と、を有し、
前記処理部は、前記第1の受光部から出力された検知信号から前方散乱光の集束位置を特定し、この集束位置から、前記第2の受光部から出力された受光信号を補正するための補正係数を求め、この補正係数を用いて前記受光信号を補正することを特徴とする蛍光検出装置。 - 前記処理部は、測定対象物に照射されるレーザ光の光強度分布の情報を用いて集束位置と受光信号を補正するための補正係数とを関係付けた補正テーブルを用いて、前記補正係数を求める請求項1に記載の蛍光検出装置。
- 前記流路と前記第1の受光部との間に、レーザ光の直接光が前記第1の受光部に照射されないように、前記光軸近傍のレーザ光を遮蔽する遮蔽板が設けられる請求項1または2に記載の蛍光検出装置。
- さらに、流路中に測定対象物を順次複数流し、補正された前記受光信号を用いて、蛍光強度の頻度分布を作成する分析部を備える請求項1~3のいずれか1項に記載の蛍光検出装置。
- さらに、前記第1の受光部にて生成される前記前方散乱光の散乱光信号の強度と前記前方散乱光の集束位置とを用いて測定対象物のサイズを求める分析部を備える請求項1~4のいずれか1項に記載の蛍光検出装置。
- 流路中を流れる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、
測定対象物が流路中の測定点を通過するように測定対象物を流して、測定対象物に対してレーザ光を照射するステップと、
測定対象物が測定点を通過するとき、レーザ光の前方散乱光が光学系を通して集光する集束位置を検知する検知信号を生成するステップと、
レーザ光の照射された測定対象物の蛍光を、集光レンズを通して受光して受光信号を出力するステップと、
前記検知信号から前方散乱光の集束位置を特定し、この集束位置から、前記受光信号を補正するための補正係数を求め、この補正係数を用いて前記受光信号を補正することにより、蛍光強度を算出するステップと、を有することを特徴とする蛍光検出方法。 - 前記受光信号を補正するとき、測定対象物に照射されるレーザ光の光強度分布の情報を用いて集束位置と受光信号を補正するための補正係数とを関係付けた補正テーブルを用いて、前記補正係数を求める請求項6に記載の蛍光検出方法。
- さらに、流路中に測定対象物を順次複数流し、補正された前記受光信号を用いて、蛍光強度の頻度分布を作成するステップを有する請求項6または7に記載の蛍光検出方法。
- さらに、前記前方散乱光の散乱光信号の強度と前記前方散乱光の集束位置とを用いて測定対象物のサイズを求めるステップを有する請求項6~8のいずれか1項に記載の蛍光検出方法。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011086913A1 (ja) * | 2010-01-15 | 2011-07-21 | 三井造船株式会社 | 蛍光測定装置及び蛍光測定方法 |
JP5611493B1 (ja) * | 2012-12-03 | 2014-10-22 | 富士電機株式会社 | 粒子線成形装置 |
JP2022145773A (ja) * | 2018-09-17 | 2022-10-04 | イングラン, エルエルシー | 液体カラム内の物体からの光収集 |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG194699A1 (en) | 2011-05-12 | 2013-12-30 | Xy Llc | Uv diode laser excitation in flow cytometry |
US9746412B2 (en) | 2012-05-30 | 2017-08-29 | Iris International, Inc. | Flow cytometer |
JP6206404B2 (ja) * | 2012-06-06 | 2017-10-04 | ソニー株式会社 | 微小粒子測定装置におけるデータ補正方法及び微小粒子測定装置 |
CN103063626A (zh) * | 2012-12-13 | 2013-04-24 | 江西科技师范大学 | 一种光路自动校正的细胞激光激发检测装置及其方法 |
CN103300863B (zh) * | 2013-06-19 | 2014-09-10 | 张英泽 | 外科手术用激光尺 |
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CN108351289B (zh) * | 2015-10-28 | 2021-11-26 | 国立大学法人东京大学 | 分析装置 |
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JP7026337B2 (ja) * | 2017-08-29 | 2022-02-28 | パナソニックIpマネジメント株式会社 | 光観測装置 |
CN108489947B (zh) * | 2018-03-22 | 2021-02-09 | 深圳大学 | 一种荧光寿命的测量方法及装置 |
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JP7260308B2 (ja) * | 2019-01-24 | 2023-04-18 | リオン株式会社 | 流体中浮遊物質測定用フローセル及び粒子計数装置 |
KR102458411B1 (ko) * | 2020-11-30 | 2022-10-26 | 에스팩 주식회사 | 입자 계수 장치 및 방법 |
CN114993897B (zh) * | 2022-07-18 | 2022-11-18 | 广东省麦思科学仪器创新研究院 | 气溶胶颗粒束束宽及颗粒分布的检测装置、套装和方法 |
WO2024194666A1 (en) * | 2023-03-21 | 2024-09-26 | Bit Group France | Method of fluorescent dye variation compensation by free state fluorescence measurement |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6129738A (ja) * | 1984-07-20 | 1986-02-10 | Canon Inc | 粒子解析装置及び粒子解析方法 |
JPS61128140A (ja) * | 1984-11-27 | 1986-06-16 | Canon Inc | 粒子解析装置 |
JPH03221837A (ja) * | 1990-01-26 | 1991-09-30 | Canon Inc | 検体測定方法及び検体測定装置 |
JPH0996603A (ja) * | 1995-09-29 | 1997-04-08 | Sumitomo Electric Ind Ltd | 流動細胞分析装置 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4643566A (en) | 1984-07-20 | 1987-02-17 | Canon Kabushiki Kaisha | Particle analyzing apparatus |
DE69118429T2 (de) | 1990-01-26 | 1996-09-12 | Canon Kk | Verfahren zur Messung einer Spezies unter Verwendung von Fluoreszenzlicht |
US5315122A (en) * | 1992-08-25 | 1994-05-24 | Becton, Dickinson And Company | Apparatus and method for fluorescent lifetime measurement |
JP2004001569A (ja) * | 2003-08-22 | 2004-01-08 | Citizen Watch Co Ltd | プリントギャップ調整機構 |
JP4384064B2 (ja) * | 2005-02-15 | 2009-12-16 | 三井造船株式会社 | 強度変調したレーザ光による蛍光検出装置 |
-
2009
- 2009-02-04 EP EP09709367A patent/EP2273253A1/en not_active Withdrawn
- 2009-02-04 US US12/866,265 patent/US8049185B2/en not_active Expired - Fee Related
- 2009-02-04 CN CN2009801043247A patent/CN101939633B/zh not_active Expired - Fee Related
- 2009-02-04 WO PCT/JP2009/000424 patent/WO2009098868A1/ja active Application Filing
- 2009-02-04 KR KR1020107018820A patent/KR101163197B1/ko not_active IP Right Cessation
- 2009-02-04 JP JP2009523506A patent/JP4489146B2/ja not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6129738A (ja) * | 1984-07-20 | 1986-02-10 | Canon Inc | 粒子解析装置及び粒子解析方法 |
JPS61128140A (ja) * | 1984-11-27 | 1986-06-16 | Canon Inc | 粒子解析装置 |
JPH03221837A (ja) * | 1990-01-26 | 1991-09-30 | Canon Inc | 検体測定方法及び検体測定装置 |
JPH0996603A (ja) * | 1995-09-29 | 1997-04-08 | Sumitomo Electric Ind Ltd | 流動細胞分析装置 |
Non-Patent Citations (1)
Title |
---|
YAMAZAKI I. ET AL.: "Laser ni yoru Bisho Ryushi Bunseki Gijutsu no Kairyo", TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS, SERIES C, vol. 60, no. 573, May 1994 (1994-05-01), pages 122 - 127, XP008140797 * |
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JP4980490B2 (ja) * | 2010-01-15 | 2012-07-18 | 三井造船株式会社 | 蛍光測定装置及び蛍光測定方法 |
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Also Published As
Publication number | Publication date |
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EP2273253A1 (en) | 2011-01-12 |
JPWO2009098868A1 (ja) | 2011-05-26 |
KR101163197B1 (ko) | 2012-07-06 |
CN101939633A (zh) | 2011-01-05 |
US8049185B2 (en) | 2011-11-01 |
KR20100115774A (ko) | 2010-10-28 |
JP4489146B2 (ja) | 2010-06-23 |
US20100314557A1 (en) | 2010-12-16 |
CN101939633B (zh) | 2012-10-31 |
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