Particle analysis system and particle analysis device

Abstract

The present invention provides technology capable of individually discriminating the states of a plurality of independent particles.　Provided is a particle analysis system comprising: a flow path through which particles flow; a plurality of detection optical systems which are disposed at positions facing each other across the flow path and which have different focal planes in the flow path; and a plurality of detection units which each include a detector that detects light derived from a corresponding detection optical system.

Description

Particle analysis system and particle analysis device

[0001] The present technology relates to particle analysis systems and particle analysis devices.

[0002] Flow cytometry has been used as a conventional method for analyzing (or analyzing, and in this technology, analysis includes the concept of analyzing) particles such as cells, microorganisms, and ribosomes. The device used for this flow cytometry is called a flow cytometer (FCM). In a flow cytometer, a laser beam of a specific wavelength is irradiated onto particles flowing through a flow channel, and light such as fluorescence, forward scattered light, and side scattered light emitted from each particle is converted into an electrical signal by a detector and digitized. Statistical analysis of the results is then performed to determine the type, size, structure, etc. of each individual microparticle.

[0003] Furthermore, in recent years, as disclosed in Patent Document 1 and the like, a so-called imaging flow cytometer (IFCM) has been developed, which acquires a two-dimensional image of fluorescence emitted from particles using an image sensor.

[0004] International Publication No. 2023 / 248561

[0005] However, conventional flow cytometers or imaging flow cytometers are primarily designed to analyze or separate particles (e.g., cells or cell clumps formed by the binding of multiple cells) as a single particle, and are therefore configured to acquire images from one direction, making it difficult to individually distinguish the states of multiple independent particles.

[0006] Therefore, the main object of the present technology is to provide a technology that can individually determine the states of multiple independent particles.

[0007] The present technology provides a particle analysis system comprising: a flow path through which particles flow; a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path; and a plurality of detection units each having a detector that detects light derived from the plurality of detection optical systems.

[0008] The present technology also provides a particle analysis device including a flow path through which particles flow, a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path, and a plurality of detection units each having a detector that detects light derived from the plurality of detection optical systems.

[0009] In this specification, the term "focal plane" refers to a plane that includes the focal point of the detection optical system.

[0010] FIG. 1 is a schematic diagram showing a configuration example according to a first embodiment of a particle analysis system 100 relating to the present technology. FIG. 1 is a schematic diagram showing an example of an embodiment of a channel T formed in a substrate. FIG. 1 is a schematic diagram showing a specific example of the detection optical system 121 and the detection unit 122 of the particle analysis system 100 according to the first embodiment. FIG. 2 is a schematic diagram showing another specific example of the detection optical system 121 and the detection unit 122 of the particle analysis system 100 according to the first embodiment. FIG. 3 is a schematic diagram showing a state in which three or more cells are held and overlapped on a particle P (particularly a carrier) in the observation direction. FIG. 1 is a schematic diagram showing a configuration example according to a second embodiment of the particle analysis system 100 relating to the present technology. FIG. 2 is a hardware configuration diagram showing an example of a computer that realizes the functions of the particle analysis system 100 relating to the present technology. FIG. 3 is a schematic diagram showing an example of the arrangement of an irradiation unit 101, a detection optical system 121, and a detection unit 122 in the case of a flow cell type channel. FIG. 4 is a schematic diagram showing an example of the arrangement of an irradiation unit 101, a detection optical system 121, and a detection unit 122 in the case of a chip type channel. FIG. 5 is a schematic diagram showing an example of staining and spectroscopic images of antibody-secreting cells and antibody-bound cells. Fig. 10 is a schematic diagram showing an example of images of antibody-secreting cells and antibody-bound cells detected by two detection units 122. Fig. 11 is a schematic diagram showing a specific example of a detection optical system and a detection unit of a conventional particle analysis system.

[0011] Hereinafter, preferred embodiments for implementing the present technology will be described with reference to the drawings. The embodiments described below illustrate examples of typical embodiments of the present technology, and are not intended to narrow the scope of the present technology. The description will be given in the following order: 1. Configuration example of a particle analysis system 100 according to a first embodiment of the present technology (1) Particles P (2) Flow path T (3) Irradiation unit 101 (4) Detection optical system 121, detection unit 122, signal processing unit 123 (5) Analysis unit 102, velocity measurement unit 124 (6) Display unit 103 (7) Sorting unit 104 2. Configuration example of a particle analysis system 100 according to a second embodiment of the present technology 3. Configuration example of hardware 4. Configuration example of an example of an embodiment of a particle analysis device according to the present technology

[0012] 1. Configuration example of a particle analysis system 100 according to a first embodiment of the present technology

[0013] Fig. 1 is a schematic diagram showing a configuration example of a particle analysis system 100 according to a first embodiment of the present technology. The particle analysis system 100 shown in Fig. 1 includes at least a plurality of detection optical systems 121 and a plurality of detection units 122. Furthermore, the particle analysis system 100 may include a light source 111 and a light guide optical system 112 that configure an irradiation unit 101, a signal processing unit 123, an analysis unit 102 and a velocity measurement unit 124, a display unit 103, a fractionation unit 104, etc., as necessary.

[0014] 1, the plurality of detection optical systems 121 are arranged such that one detection optical system 121 faces the other detection optical system 121 at a position substantially on the same straight line, but the present technology is not limited to this configuration, and other configurations may also be adopted. This will be described later in "2. Configuration example according to the second embodiment of the particle analysis system 100 according to the present technology."

[0015] (1) Particle P

[0016] In the present technology, the term "particles" may broadly include biological particles such as cells, microorganisms, and ribosomes, as well as synthetic particles such as latex particles, gel particles, and industrial particles. In the present technology, the particles P are contained in a fluid such as a liquid sample S.

[0017] Bio-related particles may include chromosomes, ribosomes, mitochondria, organelles (cell organelles), and the like that make up various cells. Cells may include animal cells (e.g., blood cells), plant cells, and the like. Microorganisms may include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Bio-related particles may also include bio-related polymers such as nucleic acids, proteins, and complexes thereof. Industrial particles may be, for example, organic or inorganic polymer materials, metals, and the like. Organic polymer materials may include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials may include glass, silica, magnetic materials, and the like. Metals may include gold colloids, aluminum, and the like.

[0018] In this embodiment, the particle P may be a carrier (sometimes referred to as a "carrier") or a cell, and the carrier may hold one or more cells or a plurality of cells. For example, a biological component (e.g., a cell or a cell-derived component (e.g., a secretion)) may be held. Holding a biological component on the carrier includes, for example, a case where the biological component is captured on the carrier or a case where the biological component is encapsulated in the carrier. The carrier may be, for example, a carrier used for secretion analysis. The carrier may be an emulsion, and in this case, the biological particles may be recovered in a state contained in the emulsion. In this case, the recovered fractionated product may be an emulsion, and the dispersoid constituting the emulsion may be a biological particle contained in the emulsion containing the particles to be separated. Specific examples of the carrier include the emulsion and beads described above. The cells held on the carrier may include, for example, antibody-secreting cells and / or antibody-binding cells.

[0019] In this embodiment, the particles P may be labeled with one or more dyes, such as fluorescent dyes. In particular, cells may be stained with multiple fluorescent dyes.

[0020] In this case, examples of fluorescent dyes that can be used include Cascade Blue, Pacific Blue, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Propidium iodide (PI), Texas Red (TR), Peridinin chlorophyll protein (PerCP), Allophycocyanin (APC), 4',6-Diamidino-2-phenylindole (DAPI), Cy3, Cy5, Cy7, and Brilliant Violet (BV421).

[0021] (2) Flow path T

[0022] The flow path T is a section through which the particles P flow. Specifically, the flow path T can be configured to form a flow in which the particles P are aligned in a substantially straight line. The flow path T may be provided in advance in the particle analysis system 100, but it is also possible to install a commercially available flow path or a disposable chip provided with a flow path.

[0023] The shape of the flow channel T is not particularly limited and can be freely designed as appropriate. For example, the flow channel T is not limited to a flow channel formed in a two-dimensional or three-dimensional substrate made of plastic, glass, or the like, but a flow channel such as that used in a conventional flow cytometer can also be used.

[0024] The particle analysis system 100 may be configured so that light from the irradiation unit 101 is irradiated onto particles P contained in a fluid flowing through a flow path T. The system may also be configured so that the light irradiation point (interrogation point) is located within a flow path structure in which the flow path T is formed. Specifically, for example, the system may be configured so that the light is irradiated onto the flow path T within a chip or a flow cell.

[0025] The width, depth, cross-sectional shape, etc. of the flow channel T are not particularly limited as long as they can form a laminar flow, and can be freely designed as appropriate. For example, a microchannel with a width of 1 mm or less can also be used in the particle analysis system 100.

[0026] The method for sending particles P through the flow channel T is not particularly limited, and particles P can be passed through the flow channel T depending on the shape of the flow channel T, etc. FIG. 2 is a schematic diagram showing an example of an embodiment of a flow channel T formed in a substrate. Specifically, for example, in the case of a chip-type flow channel T (chip system) formed in a substrate shown in FIG. 2, a sample liquid containing particles P is introduced into a sample liquid flow channel T11, and sheath liquid is introduced into two sheath liquid flow channels T12a and T12b. The sample liquid flow channel T11 and the two sheath liquid flow channels T12a and T12b merge to form a main flow channel T13. The sample liquid laminar flow sent through the sample liquid flow channel T11 and the sheath liquid laminar flow sent through the two sheath liquid flow channels T12a and T12b merge in the main flow channel T13, thereby forming a core flow in which the sample liquid laminar flow is sandwiched between the sheath liquid laminar flows. In this case, a vibration element can be applied to a portion of the substrate surface, and droplets can be formed from a liquid column ejected from an orifice.

[0027] Furthermore, for example, in the case of a flow cell type flow path T (flow cell system), the sheath liquid and sample liquid are injected into, for example, a conical container (not shown). The conical container is installed with its apex pointing vertically downward, and a tube or the like for introducing the sheath liquid is connected to the upper side. The top of the conical container is open, and a vibrating element is attached and sealed with an O-ring. The sample liquid is injected vertically from above the container. The conical container narrows at the bottom and connects to a cuvette section formed inside with a linear flow path. A laminar flow is formed in the conical container, with the sheath liquid surrounding the sample liquid. When the sheath liquid continues as a laminar flow to the cuvette section, detection is performed by irradiating light in the linear flow path. A removable outlet nozzle is installed at the end of the linear flow path, and the connecting section is sloped so that it continuously narrows from the cuvette outlet to the outlet nozzle.

[0028] (3) Irradiation unit 101

[0029] The irradiation unit 101 has one or more light sources 111, and irradiates particles P contained in the fluid with light from the light sources 111. When there are multiple light sources 111, the light sources 111 may emit light of the same wavelength or light of different wavelengths.

[0030] The type of light irradiated from the irradiating unit 101 is not particularly limited, but light with a constant light direction, wavelength, and light intensity is preferred in order to reliably generate light from the particles P. Specifically, examples of such light include laser light and LED.

[0031] Examples of laser light include semiconductor lasers, argon ion (Ar) lasers, helium-neon (He—Ne) lasers, dye lasers, krypton (Cr) lasers, and solid-state lasers that combine semiconductor lasers with wavelength conversion optical elements, and these can be used alone or in combination of two or more.

[0032] When the irradiation unit 101 has a plurality of light sources 111, the irradiation unit 101 may be configured to combine light emitted from the plurality of light sources 111 and to irradiate the particles P with the combined light. In the present technology, the irradiation unit 101 is preferably configured to irradiate light from the plurality of light sources 111 at different positions in the flow direction of the fluid. In this case, the positions to which light is irradiated may be configured to irradiate at least two or more (e.g., two, three, four, five, six, or seven) spots, and the particle analysis system 100 may be configured so that the particles P pass through the spots.

[0033] The irradiation unit 101 may include a light-guiding optical system 112 for guiding light to a predetermined position. The light-guiding optical system 112 may include optical components such as a beam splitter group, a mirror group, and an optical fiber to combine multiple beams of light. The light-guiding optical system 112 may also include a lens group for focusing the combined excitation light, and may include, for example, an objective lens 1210.

[0034] In this embodiment, a single excitation light source is used to detect a common irradiation area on the flow path T using the detection unit 122 described below, but each detection unit 122 may have its own excitation light source.

[0035] In addition, in FIG. 1, light is irradiated onto particles P flowing through the flow path T (Cuvette detection method), but when a fluid is ejected as a jet flow from the orifice of the flow path T, light may be irradiated onto the liquid column of the jet flow (Jet in Air detection method).

[0036] (4) Detection optical system 121, detection unit 122, and signal processing unit 123

[0037] In the present technology, as shown in Fig. 3 , the plurality of detection optical systems 121 are arranged at positions facing each other across a flow path T through which particles P flow, and have different focal planes within the flow path T. Furthermore, as shown in Fig. 3 , the plurality of detection units 122 each have a detector that detects light derived from the plurality of detection optical systems 121.

[0038] As described above, in conventional flow cytometry or imaging flow cytometry, it is difficult to individually distinguish the staining states of multiple independent particles.

[0039] Fig. 12 is a schematic diagram showing a specific example of the detection optical system and detection unit of a conventional particle analysis system. Specifically, in "Arrangement 1" shown in Fig. 12, when a carrier passes through the detection area, as an example of particle P, it is easy to individually distinguish the staining states of cell A and cell B held on the carrier. However, when the two cells overlap in the observation direction and are distributed beyond the focal depth range, as in "Arrangement 2" shown in Fig. 12, the image becomes unclear due to defocusing, and the overlapping makes it difficult to distinguish. Furthermore, when the two cells completely overlap in the observation direction, as in "Arrangement 3" shown in Fig. 12, only an image of one of the cells can be acquired, making it impossible to distinguish them.

[0040] This technology solves this problem by providing different observation directions within the flow channel T, so that even if multiple particles P overlap in the imaging direction, the overlapping particles can be separated and the staining state can be determined from the imaging results of the different observation directions. Furthermore, it is possible to simultaneously acquire images with different focal positions for each direction, and even if the focal direction positions of each particle are distributed beyond the focal depth of the detection optical system 121, a more focused image can be acquired.

[0041] The detection optical system 121 allows light of a predetermined detection wavelength to reach a corresponding detector. The detection optical system 121 may include a spectroscopic optical system 1211 such as a prism or a diffraction grating. It may also include a wavelength separation unit such as a dichroic mirror 1213 or an optical filter. The detection optical system 121 may be configured, for example, to separate light from the particles P and detect light of different wavelength ranges in a plurality of detectors, the number of which is greater than the number of fluorescent dyes, or in different detection regions within a single detector having spatial resolution. The detection optical system 121 may also be configured, for example, to separate light from the light from the particles P that corresponds to the fluorescent wavelength range of the fluorescent dye, and detect the separated light in a corresponding detector.

[0042] The detection unit 122 detects light (hereinafter also referred to as "measurement target light") emitted from the particle P when the particle P is irradiated with light by the irradiation unit 101. In this case, examples of the light to be detected include fluorescence, scattered light (e.g., any one or more of the group consisting of forward scattered light, backward scattered light, and side scattered light), transmitted light, and reflected light.

[0043] In the present technology, the detection unit 122 is composed of at least two or more detectors, and the detectors include light-receiving elements, and may have, for example, a light-receiving element array. Specifically, the detectors may be one or more of a group consisting of a photomultiplier tube (PMT), an avalanche photodiode (APD), a multi-pixel photon counter (MPPC), an image sensor, an event-based vision sensor (EVS), and a signal-photon avalanche diode (SPAD). The PMT may include, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detector may also include an imaging element such as a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS). The light to be measured is converted into an electrical signal by the detector, and the electrical signal is output to the analysis unit 102 (described later) for acquiring information about the particle P. For details about the image sensor, EVS, and SPAD, see International Publication No. 2023 / 248561.

[0044] 3 is a schematic diagram showing a specific example of the detection optical system 121 and the detection unit 122 of the particle analysis system 100 of the first embodiment. The specific example shown in FIG. 3 shows a cross-sectional view of a flow path T. In this specific example, a plurality of detection optical systems 121 (in the example shown in FIG. 3, these are objective lenses 1210 and band-pass filters 1212) are provided at positions facing each other across the flow path T, which are used to detect carriers, i.e., particles P. By using the band-pass filters 1212, an image using only light of a target wavelength (particularly, fluorescent wavelengths) can be obtained by the detection unit 122.

[0045] Then, the light guided from these detection optical systems 121 reaches the respective detection units 122 (image sensors in the example shown in FIG. 3), which enable these detection units 122 to simultaneously acquire multiple image data for the same particle P. As a result, even if, for example, two cells in a carrier overlap in the observation direction, the state of each particle P (particularly, the fluorescent staining state of the cells held by the particle P) can be individually determined by referring to the respective images acquired by image sensor 1 and image sensor 2 in FIG. 3.

[0046] Furthermore, as described above, the multiple detection optical systems 121 arranged at positions opposite each other across the flow channel T have different focal planes within the flow channel T. Specifically, it is preferable that the focal planes are located away from the central axis of the flow channel T in the flow direction, and that the focal planes are set at positions receding toward the detectors of each of the multiple detection optical systems. This allows image sensor 1 to acquire a more focused and clearer image of cells distributed to the left of the central axis of the flow channel T in FIG. 3 (see cell A in FIG. 3 ), and image sensor 2 to acquire a more focused and clearer image of cells distributed to the right of the central axis of the flow channel T in FIG. 3 (see cell B in FIG. 3 ), thereby further improving the ability to distinguish between each cell.

[0047] FIG. 4 is a schematic diagram showing another specific example of the detection optical system 121 and the detection unit 122 of the particle analysis system 100 of the first embodiment. In the specific example shown in FIG. 4 , the detection optical system 121 includes a spectroscopic optical system 1211, and a cross-sectional view of the flow path T is shown. In this specific example, a plurality of detection optical systems 121 (objective lenses 1210 and spectroscopic optical systems 1211 in the example shown in FIG. 3 ) are provided at positions facing each other across the flow path T, for carrying carriers, i.e., particles P. By including the spectroscopic optical system 1211 in the detection optical system 121, light containing multiple fluorescent light beams collected by the objective lens 1210 is separated and imaged on the image sensor 1 and the image sensor 2 shown in FIG. 4 as images separated for each fluorescent dye. In this way, this embodiment can also handle cells stained with multiple fluorescent dyes, and can simultaneously acquire separate spectroscopic images.

[0048] 5 is a schematic diagram showing a state in which three or more cells are held and overlapped by a particle P (particularly, a carrier) in the observation direction. When three or more cells overlap in the observation direction, the fluorescent staining state of the central cell sandwiched between them cannot be detected. Therefore, in this case, the size of the particle P (particularly, the carrier) can be appropriately adjusted to reduce the probability of three or more cells being held to an acceptable level.

[0049] The detection unit 122 may also include a signal processing unit 123 that converts the electrical signal obtained by the detector into a digital signal. The signal processing unit 123 may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit 123 may be transmitted to the analysis unit 102. The digital signal may be treated by the analysis unit 102 as data related to light (hereinafter also referred to as "light data"). The light data may be, for example, light data including fluorescence data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescence (which may include, for example, feature quantities such as area, height, and width).

[0050] (5) Analysis unit 102, speed measurement unit 124

[0051] The particle analysis system 100 according to this embodiment may include an analysis unit 102 and a velocity measurement unit 124. The analysis unit 102 analyzes the data output from the detection unit 122.

[0052] Specifically, the analysis unit 102 may include, for example, a processing unit that processes various data (e.g., optical data, etc.) and a storage unit that stores various data. When optical data corresponding to a fluorescent dye is acquired from the detection unit 122, the processing unit may perform fluorescence leakage correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye.

[0053] The fluorescence separation process may be performed according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259, for example. When the detection unit 122 includes an image sensor, the processing unit may acquire morphological information of the particles P based on an image acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing process.

[0054] In this embodiment, the particle analysis system 100 includes a sorting unit 104, which will be described later, and the analysis unit 102 can determine whether or not to sort the particles P based on the optical data and / or morphological information. Then, the analysis unit 102 controls the sorting unit 104 based on the result of the determination, and the sorting unit 104 can sort the particles P.

[0055] The analysis unit 102 may be configured to output various data (e.g., optical data, image data, etc.). For example, the analysis unit 102 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The analysis unit 102 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The display unit 103 for executing the output will be described later in "(6) Display Unit 103." The analysis unit 102 may also include an input unit (e.g., a mouse, a keyboard, an information terminal, etc.) for executing the input.

[0056] The analysis unit 102 may be configured as a general-purpose computer, for example, as an information processing device including a CPU (Central Processing Unit), RAM (Random Access Memory), and ROM (Read Only Memory). The analysis unit 102 may be included in a housing that includes the irradiation unit 101, the detection optical system 121, and the detection unit 122, or may be located outside the housing. Furthermore, the various processes or functions performed by the analysis unit 102 may be realized by a server computer or a cloud connected via a network.

[0057] The velocity measurement unit 124 measures, for example, the velocity of the particle P flowing through the flow path T relative to the velocity measurement unit 124. In this embodiment, a case is exemplified in which the velocity measurement unit 124 is stationary with respect to the flow path T, and therefore, in the following description, it is referred to as the velocity measurement unit 124 measuring the velocity of the particle P.

[0058] The velocity measurement unit 124 may employ various detection methods, such as an electrostatic method or an optical method, that can detect the velocity of the particle P. The velocity of the particle P detected by the velocity measurement unit 124 is sent to the analysis unit 102 as needed.

[0059] In this embodiment, the velocity measurement unit 124 may be omitted when the velocity of the particles P flowing through the flow path T is known, such as when the velocity of the particles P is controlled so as to be maintained at a desired velocity by controlling a pump system that delivers the liquid sample S. However, even when the velocity of the particles P is known, the velocity of the particles P may fluctuate due to changes in the ambient temperature or the resistance of the liquid delivery system, and therefore the velocity of the particles P may be actually measured using the velocity measurement unit 124.

[0060] In this embodiment, the analysis unit 102 may evaluate whether or not antibody secretion from antibody-secreting cells occurs, as will be specifically described in "Example 2" below.

[0061] (6) Display section 103

[0062] The particle analysis system 100 according to this embodiment may include a display unit 103 .

[0063] The display unit 103 displays the data output from the analysis unit 102. Specific examples of the display unit 103 include a display, a monitor, a printer, and a personal digital assistant. The display unit 103 may be included in a housing in which the irradiation unit 101, the detection optical system 121, and the detection unit 122 are provided, or may be located outside the housing. Furthermore, various processes or functions performed by the display unit 103 may be realized by a server computer or a cloud connected via a network.

[0064] The display unit 103 can display image data output from the analysis unit 102. The image data can be, for example, fluorescence image data. The image data can also include spectral image data obtained by spectral analysis using the spectroscopic optical system 1211 and acquiring the spectral image data by the detection unit 122.

[0065] Furthermore, as will be described later in "Example 2," the image data may be displayed for each detection unit 122. In other words, the user may be shown image data based on two or more detection units 122. This makes it clear which detection unit 122 has acquired the image data.

[0066] (7) Preparation section 104

[0067] The particle analysis system 100 according to this embodiment may include a fractionation unit 104 .

[0068] The sorting unit 104 includes a charging unit that charges droplets containing the particles P, and sorts out the target particles P to be sorted. Specifically, for example, droplets containing the particles P are generated by vibrations generated by a vibration element such as a piezoelectric element, and the droplets to be sorted are charged by the charging unit in response to instructions from the above-mentioned analysis unit 102, and the direction of travel of the droplets is controlled by a counter electrode. In this embodiment, the direction of travel of the particles P may be controlled within the flow path structure to perform sorting. In this case, the flow path structure may be provided with a control mechanism that uses, for example, pressure (spray or suction) or charge, and an example of the flow path structure is a chip as shown in FIG. 2.

[0069] 2. Configuration example of a second embodiment of the particle analysis system 100 according to the present technology

[0070] Fig. 6 is a schematic diagram showing a configuration example of a particle analysis system 100 according to a second embodiment of the present technology. The particle analysis system 100 shown in Fig. 6 includes at least a plurality of detection optical systems 121 and a plurality of detection units 122. Furthermore, the particle analysis system 100 may include a light source 111 and a light guiding optical system 112 that configure an irradiation unit 101, a signal processing unit 123, an analysis unit 102 and a velocity measurement unit 124, a display unit 103, a fractionation unit 104, etc., as necessary.

[0071] In addition, in Figure 6, in the first embodiment shown in Figure 3, the multiple detection optical systems 121 are arranged so that one detection optical system 121 faces the other detection optical system 121 at a position approximately on the same straight line as the other detection optical system 121, whereas the multiple detection optical systems 121 are arranged so that they face each other at different positions in the flow direction of the flow path T.

[0072] In this way, in this technology, it is not necessarily necessary to adopt a configuration in which a common irradiation area on the flow path by one light source 111 is detected by each detection unit 122 via multiple detection optical systems 121, but it is also possible to adopt a configuration in which different irradiation areas separated in the flow direction of the flow path T by multiple light sources 111 are detected by each detection unit 122 via multiple detection optical systems 121.

[0073] The other parts are as described above in "1. Configuration example of the first embodiment of the particle analysis system 100 according to the present technology," and therefore description thereof will be omitted here.

[0074] 3. Hardware configuration example

[0075] The signal processing unit 123 and the analysis unit 102 according to the above-described embodiments can be realized, for example, by a computer 1000 configured as shown in Fig. 7. Fig. 7 is a hardware configuration diagram showing an example of the computer 1000 that realizes the functions of the signal processing unit 123 and the analysis unit 102. The computer 1000 includes a CPU 1100, a RAM 1200, a ROM (Read Only Memory) 1300, a HDD (Hard Disk Drive) 1400, a communication interface 1500, and an input / output interface 1600. The components of the computer 1000 are connected by a bus 1050.

[0076] The CPU 1100 operates and controls each component based on programs stored in the ROM 1300 or the HDD 1400. For example, the CPU 1100 loads the programs stored in the ROM 1300 or the HDD 1400 into the RAM 1200 and executes processing corresponding to the various programs.

[0077] The ROM 1300 stores boot programs such as a Basic Input Output System (BIOS) executed by the CPU 1100 when the computer 1000 is started, as well as programs that depend on the hardware of the computer 1000 .

[0078] The HDD 1400 is a computer-readable recording medium that non-temporarily records programs executed by the CPU 1100 and data used by the programs. Specifically, the HDD 1400 is a recording medium that records programs for realizing various operations, which are examples of program data 1450.

[0079] The communication interface 1500 is an interface for connecting the computer 1000 to an external network 1550 (e.g., the Internet). For example, the CPU 1100 receives data from other devices and transmits data generated by the CPU 1100 to other devices via the communication interface 1500.

[0080] The input / output interface 1600 is an interface for connecting the input / output device 1650 and the computer 1000. For example, the CPU 1100 receives data from input devices such as a keyboard and a mouse via the input / output interface 1600. The CPU 1100 also transmits data to output devices such as a display, a speaker, and a printer via the input / output interface 1600. The input / output interface 1600 may also function as a media interface for reading programs and the like recorded on a predetermined recording medium. Examples of media include optical recording media such as DVDs (Digital Versatile Discs) and PDs (Phase Change Rewritable Discs), magneto-optical recording media such as MOs (Magneto-Optical Disks), tape media, magnetic recording media, and semiconductor memories.

[0081] For example, when the computer 1000 functions as the signal processing unit 123 and the analysis unit 102 according to the above-described embodiment, the CPU 1100 of the computer 1000 executes a program loaded onto the RAM 1200 to realize the functions of the signal processing unit 123 and the analysis unit 102. The HDD 1400 stores the program and the like. The CPU 1100 reads and executes the program data 1450 from the HDD 1400, but as another example, the CPU 1100 may obtain these programs from another device via an external network 1550.

[0082] 4. Configuration example of an embodiment of a particle analyzer according to the present technology

[0083] The particle analyzer according to the present technology includes at least a plurality of detection optical systems 121 and a plurality of detection units 122. The particle analyzer may also include a light source 111 and a light-guiding optical system 112 that configure an irradiation unit 101, a signal processing unit 123, an analysis unit 102 and a velocity measurement unit 124, a display unit 103, a fractionation unit 104, etc., as necessary.

[0084] The particle analysis device according to the present technology is a device configured to execute the analysis process of the particle analysis system 100 described above, and each part is as described above in "1. Configuration example of the first embodiment of the particle analysis system 100 according to the present technology" and "2. Configuration example of the second embodiment of the particle analysis system 100 according to the present technology," so description thereof will be omitted here.

[0085] The present technology can also employ the following configurations. [1] A particle analysis system comprising: a flow path through which particles flow; a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path; and a plurality of detection units each having a detector that detects light emitted from the plurality of detection optical systems. [2] The particle analysis system according to [1], wherein the focal plane is offset from a central axis of the flow path in the flow direction. [3] The particle analysis system according to [1] or [2], wherein the focal plane is set at a position receding toward the detector of each of the plurality of detection optical systems. [4] The particle analysis system according to any one of [1] to [3], wherein one of the plurality of detection optical systems is arranged on a substantially collinear line with the other detection optical system so as to face the other. [5] The particle analysis system according to any one of [1] to [3], wherein the plurality of detection optical systems are arranged at different positions in the flow direction of the flow path so as to face each other. [6] The particle analysis system according to any one of [1] to [5], wherein the detector includes one or more of the group consisting of a PMT (photomultiplier tube), an APD (avalanche photodiode), an MPPC (multi-pixel photon counter), an image sensor, an EVS (event-based vision sensor), and a SPAD (signal-photon avalanche diode). [7] The particle analysis system according to any one of [1] to [6], wherein the particle is a carrier. [8] The particle analysis system according to any one of [1] to [6], wherein the particle is a cell. [9] The particle analysis system according to [7], wherein the carrier holds one or more cells.

[10] The particle analysis system according to [7], wherein the carrier holds a plurality of cells.

[11] The particle analysis system according to

[10] , wherein the plurality of cells includes antibody-secreting cells.

[12] The particle analysis system according to

[10] or

[11] , wherein the plurality of cells includes antibody-binding cells.

[13] The particle analysis system according to [7], wherein the carrier is either an emulsion or beads.

[14] The particle analysis system according to any one of [1] to

[13] , wherein the detection optical system includes a spectroscopic optical system.

[15] The particle analysis system according to [9] or

[10] , wherein the cells are stained with a plurality of fluorescent dyes.

[16] The particle analysis system according to any one of [1] to

[15] , further comprising an analysis unit that analyzes data output from the detection unit.

[17] The particle analysis system according to

[16] , further comprising a display unit that displays data output from the analysis unit.

[18] The particle analysis system according to

[17] , wherein the display unit displays image data output from the analysis unit.

[19] The particle analysis system according to

[18] , wherein the image data is displayed for each detection unit.

[20] The particle analysis system according to any one of

[16] to

[19] , wherein the analysis unit evaluates the presence or absence of antibody secretion from antibody-secreting cells.

[21] The particle analysis system according to any one of [1] to

[20] , further comprising a sorting unit that sorts the particles of interest.

[22] A particle analysis device comprising: a flow path through which particles flow; a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path; and a plurality of detection units each having a detector that detects light derived from the plurality of detection optical systems.

[0086] The present technology will be described in more detail below based on examples. Note that the examples described below are examples of typical examples of the present technology, and the scope of the present technology should not be construed as being narrow.

[0087] Example 1: Example of light source 111 and light-guiding optical system 112

[0088] In the first embodiment, an example of a light source 111 and a light guide optical system 112 that irradiates a detection region with excitation light from the light source 111 in order to perform detection from two opposing directions with respect to the flow channel T will be described.

[0089] FIG. 8 is a schematic diagram showing an example of the arrangement of the irradiation unit 101, detection optical system 121, and detection unit 122 in the case of a flow cell type flow channel T. In the case of a flow cell type flow channel T, as shown in FIG. 8, a light source 111 can be arranged from a side surface in a direction perpendicular to the opposing observation direction to irradiate excitation light. Each detection optical system 121 then acquires a fluorescent image from the side of the irradiation. Note that, although not shown, different illumination regions separated in the flow direction of the flow channel T by multiple light sources 111 may be detected by the respective detection units 122 via multiple detection optical systems 121 to acquire fluorescent images, unlike the configuration shown in FIG. 8.

[0090] 9 is a schematic diagram showing an example of the arrangement of the irradiation unit 101, detection optical system 121, and detection unit 122 in the case of a chip-type flow channel T. In the case of a chip-type flow channel T, for example, as shown in FIG. 9 , a method of irradiating excitation light from the same direction as one of the detection optical systems 121 can be used. In FIG. 9 , excitation light is introduced via a dichroic mirror 1213 that is additionally arranged at a 45° angle to one of the detection optical systems 121, and is irradiated onto the flow channel T by an objective lens 1210 included in the detection optical system 121. Meanwhile, fluorescence from the flow channel T passes through the dichroic mirror 1213 and is detected by the detection unit 122.

[0091] In the case of a chip-type channel T, similarly to the case of a flow cell-type channel T, different irradiation areas separated in the flow direction of the channel T by a plurality of light sources 111 may be detected by respective detection units 122 via a plurality of detection optical systems 121 to acquire a fluorescent image. In addition, in the case of a chip capable of irradiating excitation light from the side (see JP 2019-184337 A), excitation light can be irradiated in the same manner as the method shown in FIG. 8 without using a dichroic mirror 1213.

[0092] Example 2: Example of detection of two cells overlapping in the observation direction

[0093] In this Example 2, we will explain the detection of two cells (e.g., an antibody-secreting cell and an antibody-binding cell (also called an "antibody-receiving cell")) that are encapsulated in a carrier particle P and overlap in the observation direction.

[0094] First, as shown in Figure 10, discriminative staining is performed to identify the cell type. Specifically, staining is performed for a specific receptor common to antibody-secreting cells and antibody-binding cells. Antibody-secreting cells are stained with fluorescent dye G (green), and antibody-binding cells are stained with fluorescent dye Y (yellow).

[0095] In this Example 2, for example, antibody-secreting cells are stained with fluorescent dye G and antibody-binding cells are stained with fluorescent dye Y, but the present technology is not limited to this. The same applies to the fluorescent dye R (red), which will be described later.

[0096] Next, as shown in Fig. 10 , discriminative staining is performed to identify antibody secretion from the cells or antibody binding on the cell surface. Specifically, in the case of antibody-secreting cells, the antibodies secreted by the cells and attached to the surface are stained with fluorescent dye R (red). Here, because some antibody-secreting cells secrete antibodies and others do not, antibody-secreting cells are stained with fluorescent dye R. Therefore, when spectral images are acquired using the spectroscopic optical system 1211, spectral images G and R are detected for antibody-secreting cells that secrete antibodies, as shown in Fig. 10 , and only spectral image G is detected for antibody-secreting cells that do not secrete antibodies.

[0097] On the other hand, in the case of antibody-bound cells, the antibody bound to the surface is stained with fluorescent dye R. Here, even among antibody-bound cells, there are those that are antibody-bound and those that are not, and therefore the antibody-bound cells are stained with fluorescent dye R. Therefore, when a spectral image is acquired using the spectroscopic optical system 1211, the spectral image Y and the spectral image R are detected for antibody-bound cells as shown in FIG. 10 , and only the spectral image Y is detected for antibody-bound cells that are not antibody-bound.

[0098] This Example 2 shows a case where these two cells are observed overlapping in the observation direction. For example, in the case of pattern 1, where there is no antibody secretion from the antibody-secreting cell, the left-hand detector 122 in FIG. 11 detects a spectral image G of the antibody-secreting cell and a spectral image Y of the antibody-bound cell overlapping in the observation direction. The right-hand detector 122 detects a spectral image Y of the antibody-bound cell and a spectral image G of the antibody-secreting cell overlapping in the observation direction. Then, a composite image in which images of each fluorescence wavelength (here, fluorescence G, fluorescence Y, and fluorescence R) are color-coded and superimposed, as shown in FIG. 11 , is created for each detector 122 and presented to the user as image data. That is, the image data may be displayed for each detector 122 on the display unit 103.

[0099] Pattern 2 shows an example in which antibody secretion occurs from antibody-secreting cells but antibody binding does not occur in antibody-binding cells. That is, the left-side detector 122 in Fig. 11 detects a spectral image G of antibody-secreting cells, a spectral image R in the event of antibody secretion from antibody-secreting cells, and a spectral image Y of antibody-bound cells that overlap in the observation direction. The right-side detector 122 detects a spectral image Y of antibody-bound cells, a spectral image G of antibody-secreting cells that overlap in the observation direction, and a spectral image R in the event of antibody secretion from antibody-secreting cells that overlap in the observation direction. Then, as in pattern 1, the user is presented with a composite image in which the images for each fluorescent wavelength are superimposed and color-coded, as shown in Fig. 11 .

[0100] Pattern 3 shows an example in which antibody secretion occurs from antibody-secreting cells and antibody binding occurs in antibody-binding cells. That is, the left-side detector 122 in Fig. 11 detects a spectral image G of antibody-secreting cells, a spectral image R in the event of antibody secretion from antibody-secreting cells, a spectral image Y of antibody-bound cells overlapping in the observation direction, and a spectral image R in the event of antibody binding in antibody-bound cells overlapping in the observation direction. The right-side detector 122 detects a spectral image Y of antibody-bound cells, a spectral image R in the event of antibody binding in antibody-bound cells, a spectral image G of antibody-secreting cells overlapping in the observation direction, and a spectral image R in the event of antibody secretion from antibody-secreting cells overlapping in the observation direction. Then, as in patterns 1 and 2, the user is presented with a composite image in which the images for each fluorescent wavelength are superimposed and color-coded, as shown in Fig. 11 .

[0101] Furthermore, in this Example 2, in addition to making a determination by a user's observation of image data, the presence or absence of antibody secretion from antibody-secreting cells may be automatically evaluated by the analysis unit 102. Furthermore, when the target particles P (e.g., carriers containing cells in a target fluorescent dye state) are sorted by the sorting unit 104, for example, six types of images with different fluorescent wavelengths and detection directions may be used as the objects of discrimination, and the results of individual analysis of each image may be integrated by the analysis unit 102 to determine whether or not to sort.

[0102] 100: Particle analysis system 101: Irradiation unit 111: Light source 112: Light guide optical system 121: Detection optical system 1210: Objective lens 1211: Spectroscopic optical system 1212: Band-pass filter 1213: Dichroic mirror 122: Detection unit 123: Signal processing unit 124: Speed ​​measurement unit 102: Analysis unit 103: Display unit 104: Analysis unit P: Particle T: Flow path S: Liquid sample

Claims

1. A particle analysis system comprising: a flow path through which particles flow; a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path; and a plurality of detection units each having a detector that detects light emitted from the plurality of detection optical systems.

2. The particle analysis system of claim 1, wherein the focal plane is offset from a flow-direction central axis of the flow channel.

3. A particle analysis system according to claim 1, wherein the focal plane is set at a position receding toward the detector side of each of the plurality of detection optical systems.

4. The particle analysis system according to claim 1, wherein one of said plurality of detection optical systems is positioned substantially collinearly with another detection optical system so as to face the other detection optical system.

5. The particle analysis system according to claim 1, wherein the plurality of detection optical systems are arranged opposite each other at different positions in the flow direction of the flow channel.

6. The particle analysis system of claim 1, wherein the detector includes any one or more of the group consisting of a PMT (photomultiplier tube), an APD (avalanche photodiode), an MPPC (multi-pixel photon counter), an image sensor, an EVS (event-based vision sensor), and a SPAD (signal-photon avalanche diode).

7. The particle analysis system of claim 1, wherein the particle is a carrier.

8. The particle analysis system of claim 1, wherein the particles are cells.

9. The particle analysis system of claim 7, wherein the carrier holds one or more cells.

10. The particle analysis system of claim 7, wherein the carrier holds a plurality of cells.

11. The particle analysis system of claim 7, wherein the carrier is one of an emulsion and beads.

12. The particle analysis system of claim 1, wherein the detection optics includes spectroscopic optics.

13. The particle analysis system of claim 9, wherein the cells are stained with multiple fluorescent dyes.

14. The particle analysis system according to claim 1, further comprising an analysis unit that analyzes data output from the detection unit.

15. The particle analysis system according to claim 14, further comprising a display unit for displaying data output from said analysis unit.

16. The particle analysis system according to claim 15, wherein the display unit displays image data output from the analysis unit.

17. The particle analysis system of claim 16, wherein the image data is displayed for each of the detectors.

18. The particle analysis system according to claim 17, wherein the analysis unit evaluates the presence or absence of antibody secretion from antibody-secreting cells.

19. The particle analysis system according to claim 1, further comprising a sorting section for sorting the particles of interest.

20. A particle analysis device comprising: a flow path through which particles flow; a plurality of detection optical systems arranged at opposing positions across the flow path and having different focal planes within the flow path; and a plurality of detection units each having a detector that detects light derived from the plurality of detection optical systems.

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