JP2010286381A - Flow cytometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To switch wavelengths of laser beams without causing optical axis deviation. <P>SOLUTION: In an illumination unit 12 of a flow cytometer, first to seventh semiconductor laser modules 25-31 emit light having each different wavelength. First to seventh output units 25a-31a of the first to seventh semiconductor laser modules are detachably connected to incidence side connectors 100-106 of a fiber bundle. Light from the first to seventh semiconductor laser modules 25-31 impinges on incidence units 35a-41a of the fiber bundle. Light impinging on the incidence units 35a-41a is emitted toward an incidence unit 34a of an optical fiber for illumination. Light from the fiber bundle 32 is synthesized in the optical fiber 34 for illumination, and the distribution of the quantity of light is uniformized. Light, where the distribution of the quantity of light has been uniformized, is emitted toward cell particles in a passage pipe from an emission unit 34b of the optical fiber for illumination. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a flow cytometer for analyzing biological properties such as cell particles.

  In recent years, there has been an increasing demand for flow cytometers that automatically analyze a large number of cell particles in various fields such as medicine and biology. Compared with a standard fluorescence microscope that measures fluorescence, the flow cytometer (1) can objectively measure a large number of cells in a short time (high speed and accuracy), (2) weak It can be measured even with light (high sensitivity and quantitativeness), (3) A lot of information (multi-parameter) can be obtained by one measurement, (4) Cluster analysis by target cell population is possible (5) The measurement can be performed under the same conditions (high reproducibility), and (6) high-speed sorting (sorting) of specific cells can be achieved.

  Samples that can be measured with a flow cytometer include, for example, (1) blood cells (white blood cells, red blood cells, platelets, etc.), (2) animal cells (cultured cells, isolated tissues, etc.), (3) plant cells, (4 ) Microorganisms (bacteria, protozoa, etc.), (5) marine organisms (plankton, etc.), (6) sperm, yeast, etc., (7) latex beads (such as quantitative analysis of cytokines by multiplex), etc. Are in a state of one by one and are required to be suspended in a liquid such as a sheath liquid. The measurable size is about 40 μm.

  In the flow cytometer, measurement is performed as follows. First, a large number of cell particles collected from blood or the like are stained with a fluorescent labeling reagent, and the stained cell particles are caused to flow in a sheath flow in which a sheath liquid flows. The cell particles flowing in the sheath flow are irradiated with laser light, and forward scattered light, side scattered light, and fluorescence emitted from the cell particles by the irradiation are detected by a photodetector. Then, based on the light signal detected by the photodetector, the biological properties of the cell particles are analyzed.

  Cell particles stained with a fluorescent labeling reagent exhibit various fluorescence characteristics depending on the wavelength of the irradiated laser beam. Therefore, the flow cytometer irradiates the cell particles in the sheath flow with light having different wavelengths from 2 to 4 types of laser generators (see, for example, Patent Document 1). Further, light emitted from each laser generator is synthesized on the same optical axis by an optical member such as a half mirror, and then the synthesized light is irradiated onto the cell particles.

Japanese Examined Patent Publication No. 4-73552

  In the flow cytometer, when the desired fluorescence characteristics cannot be obtained as a result of analysis or when changing the cell particles to be measured, the laser generator is used to switch the wavelength of the laser light. It is exchanged for others as appropriate. Therefore, it is necessary to adjust the optical axis between the laser generator and an optical member such as a half mirror every time the wavelength of the laser beam is switched. At that time, the position of the laser generator and the optical member had to be finely adjusted again, which took time and effort. Further, depending on the optical axis adjustment, an optical axis shift may occur. Further, when a half mirror or the like is used, there is a possibility that an optical axis shift occurs due to the passage of time.

  An object of this invention is to provide the flow cytometer which can switch the wavelength of a laser beam, without generating an optical axis shift | offset | difference.

  In order to achieve the above object, the present invention provides a flow cytometer for irradiating a liquid containing cell particles with light and analyzing the cell particles based on scattered light and fluorescence emitted from the cell particles. A plurality of optical fibers each having a plurality of light sources that emit different light, a first incident portion that receives light from the light source, and a first emission portion that emits light from the first incident portion; The incident portion is branched corresponding to each light source, the first emission portion is bundled and held in the emission side connector, and the second light into which light from the plurality of first emission portions enters. An incident portion and a second emitting portion that emits light from the second incident portion toward the cell particles in the liquid, and the second incident portion is connected via a connector receiver connected to the emitting side connector. In contact with the first emitting part. Characterized in that it comprises one illumination optical fiber to be. Here, it is preferable that the first incident portion is provided with an incident-side connector that is detachable with respect to each light source.

  According to the present invention, when switching the wavelength of light emitted from a light source, the optical fiber in the fiber bundle connected to the light source to be switched is removed, and the optical fiber in the removed fiber bundle is newly used. There is no risk of optical axis misalignment.

It is the schematic of the flow cytometer of this invention. It is the schematic of an illumination part. It is sectional drawing which shows the output part of each optical fiber in a fiber bundle. It is a graph which shows the light quantity distribution of the light emitted from the output part of each optical fiber in a fiber bundle.

  As shown in FIG. 1, a flow cytometer 10 includes a flow path forming unit 11 that forms a flow path of a suspension and sheath liquid containing cell particles such as cells stained with a fluorescent dye or a fluorescent antibody, and cell particles. Illuminating unit 12 that illuminates light L, forward-confused light detecting unit 13 that detects forward scattered light emitted from cell particles by illumination, and side scattered light and fluorescence emitted from cell particles by illumination are detected. A fluorescence / side scattered light detection unit 14 and a signal processing unit 15 that performs various processes based on a light signal detected by each light detection unit are provided.

  The flow channel forming unit 11 includes a sheath liquid supply unit 20, a suspension supply unit 21, and a flow channel tube 22. The sheath liquid supply unit 20 supplies the sheath liquid to the channel tube 22 at a constant supply speed. Thereby, a sheath flow is formed in the flow path tube 22. The suspension supply unit 21 supplies the suspension into the channel tube 22 in which the sheath flow is formed, so that the cell particles in the suspension are aligned according to the flow of the sheath flow. The flow channel tube 22 is formed of a transparent glass tube, and the light L from the illumination unit 12 is irradiated to particles in the sheath flow through the flow channel tube 22.

  As shown in FIG. 2, the illumination unit 12 includes first to seventh semiconductor laser modules 25 to 31, a fiber bundle 32, an illumination optical fiber 34, and a controller 42. The first semiconductor laser module 25 outputs light having a wavelength of 375 nm from the first output unit 25a, the second semiconductor laser module 26 outputs light having a wavelength of 405 nm from the second output unit 26a, and the third semiconductor laser module 27 Outputs light having a wavelength of 445 nm from the third output unit 27a, the fourth semiconductor laser module 28 outputs light having a wavelength of 473 nm from the fourth output unit 28a, and the fifth semiconductor laser module 29 outputs light having a wavelength of 488 nm. Is output from the fifth output unit 29a, the sixth semiconductor laser module 30 outputs light having a wavelength of 532 nm from the sixth output unit 30a, and the seventh semiconductor laser module 31 outputs light having a wavelength of 633 nm to the seventh output unit 31a. Output from. The controller 42 is connected to the first to seventh semiconductor laser modules 25 to 31 and controls the amount of light output from the first to seventh output units 25a to 31a.

  The fiber bundle 32 is composed of seven optical fibers 35 to 41 each consisting of a core and a clad, and the incident portions 35a to 41a of the respective optical fibers are the first to seventh output portions 25a to 31a of the first to seventh semiconductor laser modules. Are branched so that they can be easily mounted and removed. In addition, incident-side connectors 100 to 106 connected to the output portions 25a to 31a of the first to seventh semiconductor laser modules are provided in the incident portions 35a to 41a of the optical fibers. Further, the exit portions (core end portions 35b to 41b and clad end portions 35c to 41c shown in FIG. 3) of the fiber bundle 32 are held together by an exit side connector 43 such as a ferrule.

  As shown in FIG. 3, the seven optical fibers 35 to 41 are arranged in the output side connector 43 so that the core end portions 35b to 41b and the clad end portions 35c to 41c constituting the output portion are located on the same plane. Is held in. Since the adhesive 65 is filled between the optical fibers 35 to 41 and the ferrule 63, the optical fibers 35 to 41 are not misaligned.

  The illumination optical fiber 34 is composed of a large-diameter optical fiber having a diameter equal to or larger than that of the fiber bundle 32. The incident portion 34a of the illumination optical fiber is provided with a connector receiver 45 that is coupled to the output connector 43 of the fiber bundle. The connector receiver 45 includes a ferrule that holds the incident portion 34a. In addition, the emission portion 34 b of the illumination optical fiber is provided with a holding portion 47 that holds the emission portion 34 b toward the cell particles in the flow channel tube 22. Similarly to the connector receiver 45, the holding portion 47 is also formed of a ferrule or the like.

  Light emitted from the first to seventh semiconductor laser modules 25 to 31 is emitted from the emitting portion 34 b of the illumination optical fiber via the fiber bundle 32. By irradiating the cell particles with the light L from the emitting portion 34b, various light such as forward confused light, side confused light, and fluorescence is emitted from the cell particles.

  Further, as shown in FIG. 4, the light emitted from the fiber bundle 32, that is, the light emitted from each of the optical fibers 35 to 41 has a large amount of light in the central portions 90a to 96a in the radial direction of the fiber, and the central portions 90a to 90a. It has mountain-shaped light quantity distributions 90 to 96 in which the light quantity decreases from 96a toward the peripheral part along the radial direction. Here, in FIG. 4, the radial direction of each optical fiber 35-41 is represented by one horizontal axis for convenience of explanation. Light having such light quantity distributions 90 to 96 is combined in the illumination optical fiber 34 and the light quantity distribution is made uniform. By irradiating the cell particles with light having a uniform light amount distribution by the illumination optical fiber 34, the light uniformly strikes the entire cell particles. In addition, the light generated by the first to seventh semiconductor laser modules 25 to 31 is guided by the fiber bundle 32 and the illumination optical fiber 34 instead of the half mirror as in the prior art, thereby eliminating the optical axis deviation. Further, the entire apparatus can be reduced in size and weight, and the cost can be reduced.

  As shown in FIG. 1, the forward scattered light detection unit 13 includes a condenser lens 70 and a photodetector 71. The condensing lens 70 condenses the forward scattered light emitted from the cell particles. The light collected by the condenser lens 70 is detected by the photodetector 71. The light signal detected by the photodetector 71 is transmitted to the signal processing unit 15.

  The fluorescence / side scattered light detector 14 includes a condenser lens 73, first to fourth half mirrors 74 to 77, first to fourth band pass filters 79 to 82, and first to fifth photodetectors. 84-88. The condensing lens 73 condenses the side scattered light and the fluorescence emitted from the cell particles in the flow channel tube 22.

  The first to fourth half mirrors 74 to 77 are constituted by spectral filters. The first half mirror 74 reflects light having a wavelength of less than 505 nm out of the light collected by the condensing lens 73 toward the first bandpass filter 79, and transmits light having a wavelength of 505 nm or more. The first band pass filter 79 transmits light having a wavelength of 488 nm ± 10 nm among the light reflected by the first half mirror 74. The transmitted light is detected by the first photodetector 84, and the detected light signal is transmitted to the signal processing unit 15.

  The second half mirror 75 reflects light having a wavelength of less than 550 nm out of the light transmitted through the first half mirror 74 toward the second bandpass filter 80, and transmits light having a wavelength of 550 nm or more. The second band pass filter 80 transmits light having a wavelength of 530 nm ± 40 nm among the light reflected by the second half mirror 75. The transmitted light is detected by the second photodetector 85, and the detected light signal is transmitted to the signal processing unit 15.

  The third half mirror 76 reflects light having a wavelength of less than 600 nm out of the light transmitted through the second half mirror 75 toward the third bandpass filter 81, and transmits light having a wavelength of 600 nm or more. The third band pass filter 81 transmits light having a wavelength of 570 nm ± 40 nm among the light reflected by the third half mirror 76. The transmitted light is detected by the third photodetector 86, and the detected light signal is transmitted to the signal processing unit 15.

  The fourth half mirror 77 reflects light having a wavelength of less than 730 nm out of light transmitted through the third half mirror 76 toward the fourth bandpass filter 82, while light having a wavelength of 730 nm or more is reflected by the fifth photodetector 88. Permeate toward. The light transmitted through the fourth half mirror 77 is detected by the fifth photodetector 88. On the other hand, light having a wavelength of 680 nm ± 30 nm among the light reflected by the fourth half mirror 77 passes through the fourth bandpass filter 82. The transmitted light is detected by the fourth photodetector 87, and the detected light signal is transmitted to the signal processing unit 15.

  The signal processing unit 15 is based on the signals transmitted from the photodetector 71 in the forward scattered light detection unit 13 and the photodetectors 84 to 88 in the fluorescence / side scattered light detection unit 14, and the biological of the particles. Analyze

  Next, the operation of the present invention will be described. First, the first to seventh semiconductor laser modules 25 to 31 that emit laser beams having wavelengths of 375 nm, 405 nm, 445 nm, 473 nm, 488 nm, 532 nm, and 633 nm are installed in the illumination unit 12. And the holding part 47 of the optical fiber for illumination is installed toward the flow path tube 22. At that time, the holding portion 47 and the flow channel tube 22 are positioned so that the light L from the emitting portion 34b of the illumination optical fiber is reliably irradiated to the cell particles in the flow channel tube 22. Then, the first to seventh output portions 25a to 31a of the first to seventh semiconductor laser modules and the incident side connectors 100 to 106 of the fiber bundle are connected, and the output connector 43 of the fiber bundle and the connector receiver of the optical fiber for illumination are connected. 45 is connected.

  Then, laser beams having wavelengths of 375 nm, 405 nm, 445 nm, 473 nm, 488 nm, 532 nm, and 633 nm are emitted from the first to seventh output units 25 a to 31 a of the first to seventh semiconductor laser modules. The seven types of light output from the first to seventh output units 25 a to 31 a are incident on the incident unit 34 a of the illumination optical fiber via the fiber bundle 32. The light incident on the illumination optical fiber 34 is emitted from the emitting portion 34b after the light quantity distribution is made uniform inside. The light L emitted from the emission part 34 b is irradiated toward the cell particles in the flow channel tube 22.

  Then, by irradiating the cell particles in the channel tube 22 with seven types of light having different wavelengths, forward scattered light, side scattered light, and fluorescence are emitted from the cell particles. The forward scattered light is collected by the condenser lens 13 of the forward scattered light detection unit 13. The collected light is detected by the photodetector 71. The detected light signal is transmitted to the signal processing unit 15.

  The side scattered light and the fluorescence are collected by the condenser lens 73 of the fluorescence / side scattered light detection unit 14. Of the collected light, light having a wavelength of less than 505 nm is reflected by the first half mirror 74 and light having a wavelength of 505 nm or more is transmitted. Of the reflected light, light having a wavelength of 488 nm ± 10 nm is transmitted through the first bandpass filter 79, and the transmitted light is detected by the first photodetector 84.

  Of the light transmitted through the first half mirror 74, light having a wavelength of less than 550 nm is reflected by the second half mirror 75, and light having a wavelength of 550 nm or more is transmitted. Of the reflected light, light having a wavelength of 530 nm ± 40 nm is transmitted through the second bandpass filter 80, and the transmitted light is detected by the second photodetector 85.

  Of the light transmitted through the second half mirror 75, light having a wavelength of less than 600 nm is reflected by the third half mirror 76, and light having a wavelength of 600 nm or more is transmitted. Of the reflected light, light having a wavelength of 570 nm ± 40 nm is transmitted through the third bandpass filter 81, and the transmitted light is detected by the third photodetector 86.

  Of the light transmitted through the third half mirror 76, light having a wavelength of less than 730 nm is reflected by the fourth half mirror 77, and light having a wavelength of 730 nm or more is transmitted. The light transmitted through the fourth half mirror 77 is detected by the fifth photodetector 88. On the other hand, light having a wavelength of 680 nm ± 30 nm is transmitted through the fourth bandpass filter 82 among the light reflected by the fourth half mirror 77, and the transmitted light is detected by the fourth photodetector 87.

  The light signals detected by the first to fifth photodetectors 84 to 88 are transmitted to the signal processing unit 15. And in the signal processing part 15, based on the signal of the light transmitted from the photodetector 71 and the 1st-5th photodetectors 84-88, the biological property of a cell particle is analyzed.

  As a result of analysis, when the desired property is not obtained, or when the cell particle to be measured is changed, any one or all of the first to seventh semiconductor laser modules 25 to 31 are replaced with laser light. Switch to another semiconductor laser module with a different wavelength. At that time, the incident side connector of the fiber bundle is removed from the output part of the semiconductor laser module to be switched, and the removed incident side connector is attached to the output part of the new semiconductor laser module. Therefore, when the semiconductor laser module is switched, there is no need to adjust the optical axis as in the prior art, so there is no possibility of optical axis deviation.

DESCRIPTION OF SYMBOLS 10 Flow cytometer 12 Illumination part 25-31 1st-7th semiconductor laser module 32 Fiber bundle 34 Illumination optical fiber 34a Incident part 34b Emission part 35-41 Optical fiber 35a-41a Incident part 35b-41b (in a fiber bundle) Core end portions 35c to 41c Clad end portion 43 Output side connector 45 Connector receiver 47 Holding portion 100 to 106 Incident side connector

Claims (2)

In a flow cytometer that irradiates a liquid containing cell particles with light and analyzes the cell particles based on scattered light and fluorescence emitted from the cell particles,
A plurality of light sources that emit light having different wavelengths from each other;
The optical system includes a plurality of optical fibers each having a first incident part for incident light from the light source and a first emission part for emitting light from the first incident part. The first incident part corresponds to each light source. A fiber bundle that is branched, and the first emission part is bundled and held in the emission side connector;
A second incident part for receiving light from the plurality of first emitting parts; and a second emitting part for emitting light from the second incident part toward the cell particles in the liquid; Comprises a single illuminating optical fiber connected to the first emitting part via a connector receiver coupled to the emitting side connector.   The flow cytometer according to claim 1, wherein an incident-side connector that is detachable with respect to each light source is provided in the first incident portion.

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