Analysis system, analysis method, and program

Abstract

The present technology relates to an analysis system, an analysis method, and a program that make it possible to sort indexes of a large amount of cells within a feasible amount of time. An analysis system according to the present technology is provided with: a particle introduction unit for introducing microparticles into a sorting container for individually sorting the microparticles; an imaging unit for imaging the sorting container; and an analysis unit for analyzing an image obtained by imaging the sorting container by means of the imaging unit and identifying the position where the microparticles in the sorting container are sorted. The present technology can be applied to, for example, a flow cytometer that performs index sorting.

Description

Analysis system, analysis method, and program

[0001] The present technology relates to an analysis system, an analysis method, and a program, and in particular to an analysis system, an analysis method, and a program that enable index sorting of a large number of cells within a realistic time period.

[0002] Flow cytometry is a technique for analyzing microparticles such as cells. The device used for flow cytometry is called a flow cytometer. In a flow cytometer, laser light of a specific wavelength is irradiated onto microparticles flowing in a single file through a flow channel, and optical detection is performed in which a photodetector detects the intensity of fluorescence, forward scattered light, side scattered light, etc. emitted from each microparticle. Based on the results of optical detection, the flow cytometer determines the type, size, structure, etc. of each individual microparticle.

[0003] In recent years, so-called cell sorter-type flow cytometers have been developed that select and individually separate target microparticles from a sheath flow based on the results of optical detection performed by the flow cytometer.

[0004] When further assays, imaging, analysis, etc. are to be performed on the sorted cells, index sorting is performed, in which the sorted cells are individually dispensed into well plates and the results of optical detection by flow cytometry are linked and recorded for each cell. For example, Patent Document 1 proposes a cartridge-type cell sorter with an index sorting function. In index sorting, for example, cells sorted by the cell sorter are ejected in the order of sorting and sorted into wells one by one.

[0005] US Patent Application Publication No. 2017 / 0122861

[0006] A droplet-type cell sorter, which isolates cells into droplets and drops them into each well, can sort approximately 1,000 to 10,000 cells per second and sort the target cells into a well plate. However, with a droplet-type cell sorter, the droplet drop position varies by millimeters, making it difficult to sort cells into a well plate with densely arranged wells. Since cell sorting destinations are limited to, for example, 96-well or 384-well plates, the cell sorter's sorting capabilities are not fully utilized.

[0007] Microchannel cartridge-type cell sorters have higher accuracy in the dropping position of cells during sorting than droplet-type cell sorters, but because the well plate must be moved by one well each time cells are sorted, it is not practical to perform index sorting of more than 10,000 cells.

[0008] The present technology has been developed in light of these circumstances, and makes it possible to perform index sorting of a large number of cells within a realistic time frame.

[0009] An analysis system according to one aspect of the present technology includes a particle introduction unit that introduces microparticles into a collection container from which the microparticles are individually collected, an imaging unit that images the collection container, and an analysis unit that analyzes the image obtained by the imaging unit capturing the collection container to identify the position within the collection container from which the microparticles were collected.

[0010] An analysis method according to one aspect of the present technology includes introducing microparticles into a collection container that individually collects the microparticles, capturing an image of the collection container, and analyzing the image obtained by capturing the image of the collection container to identify the position within the collection container from which the microparticles were collected.

[0011] A program according to one aspect of the present technology causes a computer to perform a process of introducing microparticles into a collection container from which the microparticles are individually collected, analyzing an image obtained by capturing an image of the collection container, and identifying the position within the collection container from which the microparticles were collected.

[0012] In one aspect of the present technology, microparticles are introduced into a collection container that individually collects the microparticles, the collection container is imaged, and the image obtained by imaging the collection container is analyzed to identify the position within the collection container from which the microparticles were collected.

[0013] 18 is a diagram showing a schematic configuration example of a particle analysis system according to an embodiment of the present technology. FIG. 19 is a diagram showing a more specific configuration example of a particle analysis system. FIG. 19 is a diagram showing a configuration example of a cell sorter. FIG. 19 is a diagram showing a configuration example of a branching unit. FIG. 19 is a diagram showing an example of the appearance of a microwell array. FIG. 20 is a diagram showing an example of the shape of a through-hole. FIG. 21 is a diagram showing an example of the shape of a well when capturing a carrier. FIG. 21 is a diagram showing an example of the configuration of an open-type chamber. FIG. 22 is a diagram showing an example of an image obtained by an imaging unit capturing an image of a well region. FIG. 23 is a diagram showing an example of the configuration of a closed-type chamber. FIG. 24 is a diagram showing an example of a chamber when collecting microparticles. FIG. 25 is a diagram explaining a processing flow in which an information processing unit tracks the position of a microparticle and records the position of the captured microparticle. FIG. 26 is a diagram explaining a method for detecting a microparticle by an information processing unit. FIG. 27 is a diagram explaining a method for tracking the position of a microparticle by an information processing unit. FIG. 28 is a diagram explaining a method for tracking the position of a microparticle by an information processing unit when a plurality of microparticles are floating in a chamber. FIG. 29 is a block diagram showing an example of the functional configuration of an information processing unit. FIG. 29 is a flowchart explaining processing performed by a particle analysis system. FIG. 29 is a flowchart explaining details of image data analysis processing performed in step S10 of FIG. 17. FIG. 29 is a diagram explaining a method for collecting cells by vibration caused by pulsed laser irradiation. FIG. 29 is a block diagram showing an example of the configuration of computer hardware.

[0014] Hereinafter, embodiments of the present technology will be described in the following order: 1. Configuration of particle analysis system 2. Operation of particle analysis system 3. Modified examples

[0015] <<1. Configuration of Particle Analysis System>> <Configuration Example of Particle Analysis System> FIG. 1 is a diagram showing a schematic configuration example of a particle analysis system according to an embodiment of the present technology.

[0016] The particle analysis system 1 shown in Fig. 1 includes a light irradiation unit 11 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 12 that detects light generated by irradiating the biological sample S with light, and an information processing unit 13 that processes information related to the light detected by the detection unit 12. Examples of the particle analysis system 1 include a flow cytometer and an imaging cytometer. The particle analysis system 1 may also include a sorting unit 14 that sorts specific microparticles P within the biological sample. An example of the particle analysis system 1 that includes the sorting unit 14 is a cell sorter.

[0017] (Biological Sample) The biological sample S may be a liquid sample containing microparticles. The microparticles may be, for example, cells or non-cellular microparticles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of the non-cellular microparticles include extracellular vesicles, particularly exosomes and microvesicles. The microparticles may be labeled with one or more labeling substances (e.g., dyes (especially fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the particle analysis system 1 may analyze particles other than cells and non-cellular microparticles as microparticles, or may analyze carriers containing beads or the like for calibration purposes. The carriers may hold, for example, biological components (e.g., cells or cell-derived components, such as secretions). 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 also be an emulsion particle. In this specification, emulsion particles encapsulating the above-described cells or non-cellular microparticles can be considered microparticles. The dispersion medium constituting the emulsion may be appropriately selected by those skilled in the art depending on, for example, the type of emulsion particle. The carrier may also be a carrier used for secretion analysis, for example.

[0018] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which microparticles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is enveloped by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from materials such as plastic or glass.

[0019] The particle analysis system 1 is configured so that light from the light irradiation unit 11 is irradiated onto the biological sample flowing through the flow path C, particularly onto microparticles in the biological sample. The particle analysis system 1 may be configured so that the interrogation point of light on the biological sample is within the flow path structure in which the flow path C is formed, or so that the interrogation point of light is outside the flow path structure. An example of the former is a configuration in which the light is irradiated onto the flow path C within a microchip or flow cell. In the latter, the light may be irradiated onto microparticles after they have left the flow path structure (particularly its nozzle portion), such as a jet-in-air flow cytometer.

[0020] (Light Irradiation Unit) The light irradiation unit 11 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 11 may be configured to focus light irradiated from one or more different light sources to one irradiation point.

[0021] (Detection Unit) The detection unit 12 includes at least one photodetector that detects light generated by irradiating light onto the microparticles. The light to be detected is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light receiving elements, and has, for example, a light receiving element array. Each photodetector may include, as the light receiving element, one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 12 may also include an imaging element such as a CCD or CMOS. The detection unit 12 can acquire images of the microparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0022] The detection unit 12 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating microparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the microparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. The detection optical system is also configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating microparticles with light, and detect the separated light using a corresponding photodetector.

[0023] The detection unit 12 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit 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 may be transmitted to the information processing unit 13. The digital signal may be treated by the information processing unit 13 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0024] (Information Processing Unit) The information processing unit 13 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 12, 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 executes fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 12 includes an image sensor, the processing unit may acquire morphological information of microparticles based on images 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 processing.

[0025] When the particle analysis system 1 includes a sorting unit 14 (described later), the information processing unit 13 can determine whether to sort specific microparticles based on the optical data and / or morphological information. Then, the information processing unit 13 controls the sorting unit 14 based on the result of the determination, and the sorting unit 14 can sort the microparticles.

[0026] The information processing unit 13 may be configured to output various types of data (e.g., optical data and images). For example, the information processing unit 13 may output various types of data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 13 may also be configured to accept input of various types of data, such as accepting gating processing on a plot by a user. The information processing unit 13 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0027] The information processing unit 13 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 13 may be included in a housing that includes the light irradiation unit 11 and the detection unit 12, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 13 may be realized by a server computer or a cloud connected via a network.

[0028] (Sorting unit) The sorting unit 14 sorts microparticles according to the determination result by the information processing unit 13. The sorting method may be a method of generating droplets containing microparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets by electrodes. The sorting method may also be a method of controlling the direction of travel of the microparticles within a flow path structure to perform sorting. The flow path structure is provided with, for example, a control mechanism using pressure (jet or suction) or electric charge. An example of the flow path structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow path structure in which a flow path C branches into a recovery flow path and a waste flow path downstream, and specific microparticles are recovered into the recovery flow path.

[0029] In the particle analysis system 1, the sorting unit 14 selects and individually dispenses target microparticles. The particle analysis system 1 also performs index sorting, in which the selected microparticles are individually dispensed into well plates and the results of optical detection are linked and recorded for each microparticle. In index sorting, for example, microparticles selected by a cell sorter are ejected in the order of sorting and dispensed one by one into the wells.

[0030] A droplet-type cell sorter, which separates microparticles into droplets and drops them into each well, can select approximately 1,000 to 10,000 microparticles per second and sort the desired microparticles into a well plate. However, with a droplet-type cell sorter, the droplet drop position varies by millimeters, making it difficult to sort microparticles into a well plate with densely arranged wells. Since the destination for microparticle sorting is limited to, for example, a 96-well plate or a 384-well plate, the cell sorter's sorting capability is not fully utilized.

[0031] Microchannel cartridge-type cell sorters have higher accuracy in the droplet placement of microparticles during sorting than droplet-type cell sorters, but because the well plate must be moved by one well each time a microparticle is sorted, it is not practical to perform index sorting of more than 10,000 microparticles.

[0032] For example, consider sorting microparticles into a microwell array in which wells are arranged at a 100 μm pitch within a 20 mm square area. In this case, index sorting of up to approximately 40,000 microparticles can be performed. Because there is no need to move the microwell array when sorting microparticles and the time required for a single microparticle to be captured in a well is less than 10 seconds, if a cell sorter capable of high-speed microparticle sorting is used, it becomes possible to sort more than 40,000 microparticles in a realistic amount of time.

[0033] However, because it is not possible to capture the microparticles in the targeted wells, it is not possible to determine which well each microparticle was captured in, and it is not possible to link each microparticle to the results of optical detection by the flow cytometer.

[0034] This technology was conceived with the above points in mind, and makes it possible to perform index sorting of large numbers of cells within a realistic time frame.

[0035] FIG. 2 is a diagram showing a more specific example of the configuration of the particle analysis system 1.

[0036] 2, particle analysis system 1 is composed of light irradiation unit 11, detection unit 12, information processing unit 13, cell sorter 21 (sorting unit 14), microwell array 22, and imaging unit 23. Note that light irradiation unit 11, detection unit 12, information processing unit 13, and sorting unit 14 have the same configurations as those described with reference to FIG.

[0037] The cell sorter 21 is a micro-channel cartridge type cell sorter that can optically detect and sort microparticles in a substrate made of plastic, glass, etc. The cell sorter 21 has a channel C and a sorting unit 14 formed therein.

[0038] Light output from the light irradiation unit 11 is irradiated onto microparticles P flowing through the flow path C in the cell sorter 21. The detection unit 12 performs optical detection to detect the intensity of scattered light, fluorescence, etc. emitted from the microparticles P irradiated with light. The information processing unit 13 recognizes the microparticles P irradiated with light based on the results of optical detection (optical data) by the detection unit 12. When the information processing unit 13 recognizes the target microparticle P, the sorting unit 14 selects the target microparticle P from among the multiple microparticles P flowing through the flow path C in the cell sorter 21 as the microparticle to be sorted.

[0039] The cell outlet of the sorting unit 14 is connected to a chamber that holds the microwell array 22 via a tube 31. The microparticles P to be sorted are introduced into the chamber in the order in which they were sorted by the sorting unit 14 (in the order in which they were recognized by the information processing unit 13).

[0040] In this way, the cell sorter 21 can be said to be a closed-type cell sorter in which the flow path through which the microparticles to be sorted in the sorting section 14 flow is connected to an inlet section that introduces the microparticles into the chamber.

[0041] The microwell array 22 is, for example, a well plate in which wells are arranged in an array at a pitch of 100 μm within a 20 mm square area. A through-hole that functions as a suction portion for sucking microparticles P into each well is formed in the bottom surface of each well of the microwell array 22. Microparticles P introduced into the chamber are sucked by the through-hole and captured in each well. Because the through-hole of the well that captured the microparticle P is blocked by the microparticle P, subsequent microparticles P will not be captured in that well.

[0042] However, because it is not possible to control which well a microparticle P is captured in, there is no correlation between the arrangement position of each microparticle P within the microwell array 22 and the order in which each microparticle P was introduced into the chamber (the order in which they were sorted by the sorting unit 14). The microwell array 22 and the chamber function as sorting containers that individually sort and hold the introduced microparticles P.

[0043] The imaging unit 23 includes pixels arranged in a two-dimensional grid pattern, for example. The imaging unit 23 may be configured with a frame-type image sensor that scans all pixels at a predetermined frame rate and outputs image data (also referred to as frame data), or an event-based vision sensor (EVS) in which event pixels that detect events based on changes in the luminance of incident light are arranged in a two-dimensional grid pattern. Note that the EVS may output event data, instead of frame data, that includes position information (X address and Y address) of the pixel that detected the event, polarity information of the detected event (positive event / negative event), and information about the time the event was detected (timestamp).

[0044] The imaging unit 23 takes an image of the inside of the chamber and supplies the image data obtained to the information processing unit 13 .

[0045] The information processing unit 13 identifies each microparticle P in the chamber using a sorting number that indicates the order in which the microparticle P was introduced into the chamber (the order in which it was sorted by the sorting unit 14), and then tracks the position of each microparticle P from the time it was introduced into the chamber until it was captured in the well based on image data supplied from the imaging unit 23.

[0046] The information processing unit 13 links and records position information indicating the position where each microparticle P on the microwell array 22 was captured (sorted) with the sorting number of that microparticle P. The information processing unit 13 also links and records the position information of each captured microparticle P with optical data about that microparticle P (such as the results of optical detection) with the sorting number.

[0047] When additional processes such as imaging, culturing, reagent reaction assays, etc. are performed on the microparticles on the microwell array 22, or adding barcode oligonucleotides as preprocessing for genetic analysis, the information processing unit 13 records these results in association with the positional information of each microparticle P after capture.

[0048] Each component of the particle analysis system 1 will be described in detail below.

[0049] <Cell Sorter> This section describes a cell sorter that selects target cells from a plurality of microparticle samples. Generally, cell sorters are widely used that convert individual microparticles into droplets outside the device, impart a certain positive or negative charge to the droplets containing the target cells, deflect them in a strong electric field, and sort the target cells into a tube positioned where the droplets fall. Meanwhile, a microchannel cartridge-type cell sorter capable of optically detecting and selecting microparticles within a substrate such as plastic or glass is suitable as the cell sorter 21 of the particle analysis system 1 of the present technology.

[0050] In this technology, the microparticles must be introduced into the chamber in the order in which they were sorted in the cell sorter 21. In a microchannel cartridge-type cell sorter, if the cell outlet and the inlet of the chamber are connected by a tube or the like, the microparticles are handled in a closed space, and the microparticles can be easily guided into the chamber in the order in which they were sorted in the cell sorter 21.

[0051] The microchannel cartridge type cell sorter selects the target microparticles (or microparticles to be discarded) recognized by the information processing unit 13 by manipulating the liquid flow path, for example, by pushing and pulling the channel branching section using an actuator or by opening and closing a valve.

[0052] Due to instability caused by disturbances in the liquid flow, the sorting speed of a microchannel cartridge-type cell sorter is slower than that of a droplet-type cell sorter, which uses a piezoelectric vibrating element to stably form droplets at a high frequency of about 100 kHz and then isolates and handles the individual microparticles contained within the droplets. However, when index sorting is performed, this is not a problem, since it is sufficient to separate (sort) less than 1% of the entire microparticle sample.

[0053] On the other hand, because there is no process of landing droplets of microparticles on the liquid surface in the tube at a speed of 10 m / s or more, the damage to microparticles in a microchannel cartridge-type cell sorter is less than that in a droplet-type cell sorter. Therefore, the microchannel cartridge-type cell sorter is suitable for applications such as culturing sorted cells.

[0054] FIG. 3 is a diagram showing an example of the configuration of the cell sorter 21.

[0055] As shown in Figure 3, in the cell sorter 21, an inlet 101, a flow path 102, an inlet 103, a flow path 104, a flow path 105, an optical detection region 106, a branching section 107, a flow path 108, a cell capture chamber 109, an outlet 110, and a cell removal port 111 are formed on a substrate 100.

[0056] In the cell sorter 21, a sample liquid containing microparticles is injected from an inlet 101. After being injected from an inlet 103, sheath liquid is branched into two in a channel 104, and each is controlled by a pump to flow through the channel inside the cell sorter 21 at a constant flow rate. In a channel 105, the sample liquid meets, sandwiched between sheath liquids, to form a core flow in the center of the channel. The channel width in the optical detection region 106 is 200 μm, and the microparticles are present in the central core flow, which is 20 μm or less in width.

[0057] In the optical detection region 106, for example, laser light of three wavelengths is irradiated, and the forward scattered light (FSC), back scattered light (BSC) from the microparticles, and multi-wavelength fluorescent signals based on the label of the sample are detected by the detection unit 12.

[0058] Fig. 4 is a diagram showing an example of the configuration of the branching section 107. Fig. 4A shows a top view of the branching section 107, and Fig. 4B shows a perspective view of the branching section 107.

[0059] Of the microparticles flowing through the flow path 105 after being irradiated with light, the target cells flow linearly through the orifice portion 121, while the other cells pass through the flow paths 108 that branch off to the left and right of the orifice portion 121 and are discharged as waste liquid from the outlet 110.

[0060] As shown in Figure 4B, a buffer solution constantly flows in flow channel 122, which is arranged perpendicular to flow channel 105, and the buffer solution constantly flows from diamond-shaped cell capture chamber 109 in the direction of cell outlet 111. The buffer solution flows in the direction of cell outlet 111 and also in the direction of flow channel 105. The buffer solution forms a flow (block flow) in the opposite direction to the traveling direction of the microparticles, so that under normal circumstances, the microparticles do not travel in the direction of cell capture chamber 109 or cell outlet 111.

[0061] When the target microparticle approaches the vicinity of the orifice portion 121, the cell capture chamber 109 is pulled up by the piezoelectric element located directly above, and the microparticle near the orifice portion 121 is drawn into the cell capture chamber 109 simultaneously with the block flow.

[0062] The width of the orifice 121 can be changed depending on the diameter of the microparticles. A narrower width of the orifice 121 allows the flow rate required to draw in the microparticles to be achieved with less up-and-down movement of the cell capture chamber 109, resulting in a faster sorting speed. Therefore, it is desirable to narrow the width of the orifice 121 to a level that allows the microparticles to pass through, taking into account the particle diameter and core flow width (particle presence range). For example, if the diameter of the microparticles is 15 μm or less, the width of the orifice 121 can be set to 30 μm, and if the diameter of the microparticles is approximately 50 μm, the width of the orifice 121 can be set to 70 μm.

[0063] However, when index sorting is performed, as described above, the sorting speed does not need to be the top priority, so the width of the orifice portion 121 may be set to about 100 μm, taking into consideration clogging of the orifice, etc.

[0064] The detection target of the detection unit 12 is not limited to scattered light or fluorescence generated by laser irradiation. For example, the detection unit 12 may perform electrical detection or cell morphology imaging.

[0065] <Microwell Array> (1) Size FIG. 5 is a diagram showing an example of the appearance of the microwell array 22.

[0066] 5, the microwell array 22 is configured by arranging a plurality of wells 151 in an array on a well substrate. A through-hole 152 is formed in the bottom surface of each well 151.

[0067] The microwell array 22 is designed to have a higher surface density of wells than conventional 96-well plates (well pitch 9.0 mm) or 384-well plates (well pitch 4.5 mm). For example, the microwell array 22 is designed to reduce the wells 151 to a size equivalent to that of microparticles and narrow the pitch between the wells 151, thereby enabling index sorting of as many cells as possible.

[0068] When cells as microparticles are captured in each well 151, the diameter of the well 151 is set to approximately 10 to 30 μm. When carriers holding cells are captured as microparticles in each well 151, the particle diameter of the carriers is expected to be approximately 30 to 80 μm, so the diameter of the well 151 is set to 40 to 90 μm. Here, the pitch between the wells 151 can be narrowed to approximately 50 to 100 μm.

[0069] In particle analysis system 1 of the present technology, the well region, which is the region in which wells 151 are formed in microwell array 22, and the outlet port, which is the portion through which microparticles are introduced into the chamber, must be included within the imaging range of imaging unit 23. For example, if the well region is a 12 mm square area and the pitch between wells 151 is 60 μm, 40,000 wells 151 can be arranged within the imaging range of imaging unit 23. Therefore, microwell array 22 can capture a larger number of microparticles than conventional well plates.

[0070] (2) Well Structure As described above, a through-hole 152 for attracting and guiding cells is formed in the bottom surface of each well 151 so that only one microparticle is captured per well 151.

[0071] When microparticles are introduced into a well plate without through-holes, the destination of the microparticles is determined by probability theory, so cells are not necessarily captured in all wells. For example, there is a possibility that the microparticles may fall outside the wells, or that multiple microparticles may be captured in one well.

[0072] By forming through-holes 152 in the bottom surface of well 151 that are large enough to prevent microparticles from passing through, the microparticles are guided to well 151 along with the flow of buffer solution in the chamber. Furthermore, when a microparticle is captured in well 151, the microparticle blocks through-hole 152 and blocks the flow of buffer solution, preventing other microparticles from being guided to well 151 where a microparticle has already been captured.

[0073] In this way, the microparticles introduced into the chamber are guided to the wells 151 whose through-holes 152 are not yet blocked, so that, in principle, one cell is captured in each of the wells 151 .

[0074] FIG. 6 is a diagram showing an example of the shape of the through-hole 152. As shown in FIG.

[0075] 6A, for example, a through-hole 152 having a circular shape when the microwell array 22 is viewed from above is formed in the bottom surface of the well 151. Also, as shown in FIG. 6B, for example, a through-hole 152 having a rectangular shape when the microwell array 22 is viewed from above is formed in the bottom surface of the well 151.

[0076] (2-1) When a cell is captured directly When a cell is captured directly, the through-hole 152 must be formed sufficiently smaller than the cell. That is, if the cell diameter is Dc and the diameter of the through-hole 152 is Dh, then Dh << Dc must be satisfied. Furthermore, if the shape of the through-hole 152 is rectangular, then if the length of the short side of the through-hole 152 is Wh, then Wh << Dc must be satisfied.

[0077] If the diameter of the cells is 10 μm or less or if the cells are easily deformed, it may not be possible to prevent the cells from passing through even a 5 μm diameter through-hole 152. However, if the diameter of the through-hole 152 is reduced to 2 to 3 μm, the flow resistance increases, which may stop the flow of the buffer solution.

[0078] Therefore, it is desirable to form a rectangular through-hole 152 with a short side length of 3 μm and a long side length of 10 μm, for example, to prevent the passage of cells while ensuring an opening area. The total opening area may be ensured by forming multiple through-holes 152 with a diameter of 3 μm in one well 151.

[0079] In this way, when cells are captured directly in wells 151, through-holes 152 must be formed with a very small diameter of 5 μm or less, and in order to ensure strength, the bottom of well 151 must have a certain thickness. Producing microwell array 22 requires highly difficult, high-aspect-ratio microfabrication technology, which results in reduced productivity and increased costs.

[0080] (2-2) When Capturing Carriers On the other hand, when capturing carriers that are slightly larger than cells, the difficulty of producing the microwell array 22 is reduced, which is expected to result in improved productivity and cost. It is desirable for the carrier to be formed using a material that is almost undeformable. When the carrier is almost undeformable, as shown in Figure 7A, if the diameter of the carrier P12 is Dn, then the passage of the carrier can be blocked by through-holes 152 such that Dh < Dn or Wh < Dn.

[0081] For example, if the particle diameter of the carrier P12 is 50 μm, the diameter of the through-holes 152 may be 40 μm. Since there is no problem with increasing the size of the through-holes 152, the microwell array 22 can be easily fabricated. Furthermore, because there is increased freedom in design, the pressure loss across the entire microwell array 22 can be appropriately adjusted.

[0082] 7B, instead of forming the through-hole 152 in a part of the bottom surface of the well 151, the through-hole 152 formed in the well substrate may function as the well 151. In this case, the through-hole 152 (well 151) has a tapered shape in cross section.

[0083] If the diameter of the top opening of well 151 is Ds and the diameter of the bottom opening is Db, then the respective diameters should satisfy the relationship Ds > Dn > Db. For example, when Dn = 50 μm, if Ds = 60 μm and Db = 40 μm, the desired particle blocking effect will be achieved. When the through-hole depth H is 80 μm, the taper angle θ is 7°. This taper angle can be added by general machining or lithography, making it possible to produce microwell arrays 22 using a simpler process.

[0084] Even when cells themselves are captured instead of carriers, a microwell array 22 can be used in which through-holes 152 that have a tapered shape in cross section function as wells 151 .

[0085] (3) Well Substrate As the material for the well substrate, a material suitable for the processing method of the well 151 is selected from glass, plastic resin, PDMS (polydimethylsiloxane), UV resin, and the like.

[0086] Possible methods for processing the well 151 include mechanical processing, laser drilling, photolithography, 3D printing, injection molding or other transfer processes, or a processing method that combines these methods.

[0087] It is desirable that the well substrate be transparent. In particular, when the imaging unit 23 images the microwell array 22 from the bottom side of the well substrate (the bottom side of the wells 151), the well substrate must be made of a transparent material. Furthermore, when fluorescent observation of cells or cell secretions within the wells 151 is performed, it is desirable that the well substrate be made of a material with sufficiently low autofluorescence. For example, the well substrate is made of COP (cycloolefin polymer) or COC (cycloolefin copolymer).

[0088] <Chamber> (1) Structure The chamber is configured as an open-type chamber in which the top surface of the microwell array 22 is open, or a closed-type chamber in which the top surface of the microwell array 22 is sealed.

[0089] FIG. 8 is a diagram showing an example of the configuration of the open-type chamber 201.

[0090] 8, a microwell array 22 is assembled inside chamber 201, and microwell array 22 divides the internal space of chamber 201 into two spaces: an upper space 231 and a lower space 232. In microwell array 22, the openings of wells 151 are formed on the upper space 231 side, and through-holes 152 are formed on the lower space 232 side.

[0091] A cell sorter connection port 211 formed on the outside of the chamber 201 is connected to a flow path formed in a resin sheet 212 with a flow path, and the outlet of the flow path is connected to the upper space 231 as a cell discharge port 221.

[0092] Furthermore, a reagent injection port 214 formed on the outside of the chamber 201 is connected to a flow channel formed in the resin sheet 215 with a flow channel, and the outlet of the flow channel is connected to the upper space 231. The reagent injection port 214 is used to adjust the height of the liquid surface in the upper space 231, control the flow rate of the microparticles P in the upper space 231, and inject a reagent for assay that is performed after the microparticles P are captured.

[0093] The upper space 231 and the lower space 232 are filled with, for example, a buffer solution injected through the reagent injection port 214. Microparticles P introduced through the cell sorter connection port 211 pass through the flow paths in the resin sheet 212 with flow paths and are discharged from the cell discharge port 221 into the upper space 231, where they move along with the flow of the buffer solution in the upper space 231 and are captured in the well 151. The cell discharge port 221 functions as a particle introduction section that introduces microparticles into the upper space 231 of the chamber 201.

[0094] A waste liquid tube 216 is connected to the lower space 232, and the buffer liquid filling the lower space 232 is discharged from the inside of the chamber 201 via the waste liquid tube 216. When the buffer liquid filling the lower space 232 is discharged through the waste liquid tube, a flow is formed for sucking the microparticles P into the well 151. A flow control pinch valve 217 is connected to the waste liquid tube 216, and functions as a mechanism for adjusting the discharge speed of the buffer liquid to set the falling speed of the microparticles P to an appropriate speed.

[0095] The upper space 231 is a box-shaped space with an open top. Because the upper space 231 is open, specific cells can be extracted from the upper side of the chamber 201 using a mechanical method such as a glass capillary. As indicated by the white arrow in Figure 8, the imaging unit 23 can image the well region of the microwell array 22 and the cell discharge port 221 from the upper space 231 side. If the chamber 201 or the microwell array 22 is made of a transparent material, the imaging unit 23 can also image the well region of the microwell array 22 and the cell discharge port 221 from the lower space 232 side.

[0096] FIG. 9 is a diagram showing an example of an image obtained by the imaging unit 23 capturing an image of the well region.

[0097] As shown in the upper, middle, and lower rows of FIG. 9, the state in which Jurkat cells as microparticles P are sucked into wells 151 and finally captured is captured by imaging unit 23 .

[0098] Here, Jurkat cells in the upper space 231 are observed from the lower space 232 side with an inverted microscope using a x10 (NA = 0.25) objective lens. The positions of the wells 151 are identified, for example, by bright-field observation, and the Jurkat cells are identified, for example, by fluorescent observation. The dimensions of each well 151 are 20 μm square and 20 μm deep, and the pitch between the wells 151 is 60 μm. A through-hole 152 with a 5 μm x 10 μm opening is formed in the 15 μm-thick bottom of each well 151. The diameter of the Jurkat cells is an average of 10 μm.

[0099] The image in Figure 9 is focused on Jurkat cells floating in the upper space 231 because the objective lens has an NA of 0.25 and a shallow depth of focus (5 to 10 μm). However, in implementing this technology, it is necessary to clearly capture images of the Jurkat cells from when they are discharged into the chamber 201 until they are captured in the well 151. Therefore, it is desirable to use a low-magnification, low-NA lens to ensure a sufficient depth of focus and a field of view (imaging range) that covers the entire well area. It is also desirable to form the cell discharge port 221 as close to the microwell array 22 as possible.

[0100] Fig. 10 is a diagram showing an example of the configuration of a closed-type chamber 201. In Fig. 10, the same components as those described above are denoted by the same reference numerals. Duplicate explanations will be omitted as appropriate.

[0101] As shown in Figure 10, upper space 231 is sealed, for example, by a glass lid 251. As indicated by the white arrow in Figure 10, imaging unit 23 can capture an image of the well region of microwell array 22 from the upper space 231 side. A sealed upper space 231 makes it easier to apply chamber 201 to automation of assays using flow path manipulation and collection of a large number of cells than an open upper space 231.

[0102] Outside the chamber 201, a liquid feed pump 253 is connected to the reagent inlet 214 via a valve 252. Furthermore, a valve 254 and a suction pump 255 are connected to the waste liquid tube 216 instead of the flow control pinch valve 217. Furthermore, a tube 256 communicates with the lower space 232, and a liquid feed pump 258 is connected to the tube 256 via a valve 257. These reagent flow rate adjustment mechanisms enable highly accurate control of the flow rate of the microparticles P in the upper space 231.

[0103] When the flow rate of the microparticles P in the upper space 231 is controlled, as shown by the solid arrows in Figure 10, buffer liquid is sent from the flow path in the resin sheet 215 with flow path and the tube 256 to the internal space of the chamber 201, and the buffer liquid in the internal space of the chamber 201 is discharged from the waste liquid tube 216.

[0104] It is also possible to separate the chamber 201 from the cell sorter 21 and recover the microparticles P from the cell sorter connection port 211. In this case, as shown by the solid arrow in Fig. 11 , a buffer solution is sent from the tube 256 to the internal space of the chamber 201, causing the microparticles P captured in each well 151 to float up from each well 151 and be recovered from the cell sorter connection port 211 via the flow paths in the resin sheet 212 with flow paths.

[0105] In this way, various combinations of flow paths and liquid delivery systems are conceivable depending on the purpose.

[0106] Whether the chamber 201 is an open type or a closed type, the chamber 201 is designed so that the cell discharge port 221 and the entire well area fall within the imaging range of the imaging unit 23 .

[0107] It is desirable that the cell outlet 111 of the cell sorter 21 and the cell sorter connection port 211 of the chamber 201 are connected by as short a path as possible using a tube 31 or the like so that the order of the microparticles is not changed along the path.

[0108] When microparticles pass through a tube, if the flow rate is 1 mL / min or less and the tube diameter is 1 mm or less, the liquid in the tube forms a laminar flow, and the microparticles move in a straight line while maintaining a constant position in the width direction of the tube. The flow in the tube is a Hagen-Poiseuille flow, with a central axis-symmetric flow velocity distribution, where the flow velocity is maximum at the center of the tube and zero at the tube wall.

[0109] Microparticles flow at the center of the tube at the highest speed, and the closer they are to the edge, the slower their speed becomes. Depending on the conditions, particles may overtake each other inside the tube.

[0110] The applicant conducted an experiment in which beads representing cells were flowed through a tube. The results of this experiment are shown below.

[0111] When 10 μm diameter beads, representing cells, were flowed through a tube with an inner diameter Dt of 0.25 mm and a length L of 1000 mm at a flow rate of 200 μL / min (average flow velocity of 0.068 m / s), the transit time of the beads through the tube varied by 36%. The shortest transit time was 8.0 seconds (flow velocity 0.125 m / s), while the longest transit time was 10.9 seconds (0.092 m / s).

[0112] This result means that if the time between the selection of a microparticle to be sorted by the cell sorter 21 and the selection of the next microparticle to be sorted is 2.9 seconds or less, the subsequent particle may catch up with the preceding particle, causing an overtaking between particles. Therefore, the cell sorter 21 must perform sorting with an interval of at least 3 seconds, or any target cells recognized by the information processing unit 13 during that time will be discarded without being selected for sorting.

[0113] Since the time difference Δt between the shortest and longest transit times is proportional to the tube length, for example, if the tube length is set to 50 mm under the above conditions, the time difference Δt will be improved to 0.15 seconds. In this case, even if the cell sorter 21 sorts at 0.2 second intervals, it can process five cells per second.

[0114] In this way, it is desirable to arrange the cell outlet 111 of the cell sorter 21 near the cell sorter connection port 211 of the chamber 201 and to make the length of the tube 31 as short as possible.

[0115] Furthermore, when 50 μm diameter beads, which are assumed to be carriers, were flowed under the same conditions, the passage time of the beads through the tube varied by 5%. The shortest passage time for the beads was 8.2 seconds (flow velocity 0.122 m / s), while the longest passage time was 8.6 seconds (0.116 m / s). It is believed that this result was obtained because the larger particle size reduced the degree of freedom of the bead position across the width of the tube.

[0116] From another perspective, it is thought that by selecting a tube with as small an inner diameter Dt as possible depending on the particle size D of the microparticles flowing through the tube or flow path, it is possible to reduce the variation in the flow rate of particles inside the tube and achieve faster index sorting. In particular, when a carrier that hardly deforms flows through the tube, overtaking will not occur if Dt<2D.

[0117] On the other hand, if the inner diameter D of the tube is made small, particles may clog the tube, increasing the risk of stopping the liquid flow. Therefore, it is desirable to determine the inner diameter Dt of the tube in a balanced manner within the range of 2D≦Dt<5D.

[0118] As explained above, in the process in which microparticles reach chamber 201 after sorting by cell sorter 21, a time difference Δt occurs depending on the position of the microparticles passing through tube 31, and it is desirable to make length L of tube 31 as short as possible and to reduce inner diameter Dt to the extent that the microparticles do not become clogged. Furthermore, it is thought that the time difference Δt will also decrease if the flow velocity V (flow rate Q) in tube 31 is increased (increased).

[0119] In principle, the time difference Δt will not become 0, and therefore, in the particle analysis system 1 of the present technology, a waiting time tm is set by adding a margin to the time difference Δt in order to prevent the replacement of microparticles. The cell sorter 21 does not select subsequent target microparticles as microparticles to be sorted until the waiting time tm has elapsed since selecting the target microparticles. In other words, if the target microparticles are recognized by the information processing unit 13 after the waiting time tm has elapsed since selecting the target microparticles, the cell sorter 21 selects the target microparticles as microparticles to be sorted. Therefore, even if the target microparticles are recognized by the information processing unit 13 during that period, the target microparticles are discarded. The waiting time tm is appropriately specified by the operator according to the conditions of use of the system.

[0120] (2) Flow velocity of microparticles in the upper space In the particle analysis system 1 of the present technology, it is assumed that the cell sorter 21 sorts approximately 1 to 100 microparticles per second, and a state occurs in which a plurality of microparticles that are not captured in the wells 151 float in the upper space 231. In other words, before the microparticles that have been previously introduced into the chamber 201 are sorted, the subsequent microparticles are introduced into the chamber 201. Care must be taken to ensure that the number of microparticles floating in the upper space 231 does not exceed the processing limit of the information processing unit 13 that tracks the microparticles.

[0121] Furthermore, if an excessive number of microparticles are floating in the upper space 231, the trajectories may change due to collisions between the microparticles, which may cause tracking by the information processing unit 13 to fail.

[0122] Therefore, it is desirable that the well capture capacity Nc exceeds the average number of particles sorted per unit time Ns (Nc≧Ns) so that the number of microparticles floating in the upper space 231 does not exceed a certain number. The well capture capacity Nc indicates the number of microparticles captured in the well 151 per unit time, and is calculated based on the flow rate of the microparticles floating in the upper space 231.

[0123] In this way, the speed of index sorting performed in the particle analysis system 1 of the present technology is also limited by the well capture capacity Nc.

[0124] Nc≧Ns is realized when the microparticles travel the average distance L from the cell discharge outlet 221 to each well 151 within 1 / Ns seconds. For example, when the average distance L is 20 mm and one cell is sorted per second by the cell sorter 21, the microparticles should travel at an average speed Vc=20 mm / s or greater between the cell discharge outlet 221 and each well 151. Furthermore, when the average distance L is 20 mm and 100 cells are sorted per second by the cell sorter 21, the microparticles should travel at an average speed Vc=2 m / s or greater between the cell discharge outlet 221 and each well 151.

[0125] The flow rate of the microparticles in the upper space 231 is adjusted by the flow rate of the liquid discharged from the cell outlet 111 of the cell sorter 21 and the flow rate of the waste liquid from the chamber 201 .

[0126] However, when cells are captured directly in well 151, if the falling speed into well 151 is too fast, the cells may deform and pass through through-hole 152, or may collide with the wall of well 151, increasing damage to the cells (reducing viability). Therefore, when cells are captured directly in well 151, it is desirable to slow down the flow rate of the cells in upper space 231, which imposes a certain limit on the sorting speed.

[0127] On the other hand, if the carriers are captured in the wells 151, the above concerns are largely eliminated. Because the cells are chemically bound to the carriers, even if the carriers fall into the wells 151 at high speed, there is little chance that the cells will detach from the carriers due to the impact. Therefore, by retaining the cells on the carriers, it is possible to further increase the sorting speed.

[0128] Because the size of the carrier is larger than the size of the cell, holding the cell on the carrier improves the visibility of the particle, allowing imaging unit 23 to image the well region at low magnification and with a wide field of view, which ultimately allows the number of wells 151 arranged in microwell array 22 to be increased.

[0129] <Method for Tracking the Position of Microparticles Based on Image Data> FIG. 12 is a diagram illustrating the flow of processing in which the information processing unit 13 tracks the positions of microparticles and records the positions of captured microparticles.

[0130] As shown in the first row of Figure 12, when the first microparticle P31 is introduced into chamber 201, information processing unit 13 detects that microparticle P31, identified by sorting number 1, is not captured in well 151 based on the image data obtained by imaging unit 23 capturing an image of microwell array 22.

[0131] 12, when a second microparticle P32 is introduced into chamber 201 and the first microparticle P31 is captured in well 151-1, information processing unit 13 detects that microparticle P31, identified by sorting number 1, has been captured in well 151-1, based on image data obtained by imaging unit 23 capturing an image of microwell array 22. Information processing unit 13 associates and records the sorting number (number 1) of microparticle P31 with the positional information of well 151-1 in which microparticle P31 was captured.

[0132] 12, when a second microparticle P32 is captured in well 151-8, information processing unit 13 detects that microparticle P31, identified by sorting number 2, has been captured in well 151-8, based on image data obtained by imaging unit 23 capturing an image of microwell array 22. Information processing unit 13 associates and records the sorting number (number 2) of microparticle P32 with the positional information of well 151-8 in which microparticle P32 was captured.

[0133] 12, information processing unit 13 detects, based on image data obtained by imaging unit 23 capturing an image of microwell array 22, that each of the first to ninth microparticles introduced into chamber 201 has been captured in well 151. Information processing unit 13 associates and records the sorting number (numbers 1 to 9) of each microparticle with the positional information of well 151 in which the microparticle was captured.

[0134] (1) Microparticle Detection Method The information processing unit 13 detects microparticles based on, for example, the difference between frames of images captured by the imaging unit 23 .

[0135] FIG. 13 is a diagram illustrating a method for detecting microparticles by the information processing unit 13. In FIG.

[0136] 13, it is assumed that frame images Pi1 to Pi5 are captured by the imaging unit 23 at times t1 to t5, respectively. In the example of Fig. 13, at time t1, a microparticle P51 is introduced into the chamber 201, at times t2 and t3, the microparticle P51 moves, and at time t4, the microparticle P51 is captured in the well 151-9.

[0137] When the difference between consecutive frames of images captured by the imaging unit 23 is taken, a difference image is obtained in which, for example, pixels whose brightness has increased have a pixel value of 1, pixels whose brightness has decreased have a pixel value of -1, and pixels whose brightness has not changed have a pixel value of 0. In the middle of Fig. 13, difference image Pi11 shows the difference between frame image Pi1 and frame image Pi2, difference image Pi12 shows the difference between frame image Pi2 and frame image Pi3, difference image Pi13 shows the difference between frame image Pi3 and frame image Pi4, and difference image Pi14 shows the difference between frame image Pi4 and frame image Pi5.

[0138] In the difference image of FIG. 13, the black areas indicate pixels with a pixel value of 0, the areas surrounded by white dashed lines indicate pixels with a pixel value of −1, and the white areas indicate pixels with a pixel value of 1.

[0139] The information processing unit 13 can detect that the microparticle P1 is moving from time t1 to time t2 based on the pixel values ​​of the difference image Pi11. The information processing unit 13 can detect that the microparticle P1 is moving from time t2 to time t3 based on the pixel values ​​of the difference image Pi12. The information processing unit 13 can detect that the microparticle P1 is moving from time t3 to time t4 based on the pixel values ​​of the difference image Pi13. The information processing unit 13 can detect that the microparticle P1 is stopping from time t4 to time t5, i.e., that the microparticle P1 is captured in the well 151 at time t4 based on the pixel values ​​of the difference image Pi14.

[0140] The information processing unit 13 acquires a difference image Pi21 (bottom of FIG. 13 ) that shows the difference between the frame image Pi5 and the base image obtained by the imaging unit 23 capturing an image of the microwell array 22 before the microparticles are introduced into the chamber 201. In the difference image Pi21, only the pixel corresponding to the position of the microparticle P51 captured in the well 151-9 at time t5 has a pixel value of 1, for example. Therefore, the information processing unit 13 can identify the coordinates of the well 151-9 in which the microparticle P51 is captured, based on the pixel values ​​of the difference image Pi21.

[0141] When the imaging unit 23 is configured with an EVS, a difference image showing the difference between successive frames in images captured by a frame-type image sensor is directly acquired by the imaging unit 23. Since the EVS detects only changes in luminance, the volume of image data acquired by the imaging unit 23 is reduced, and the information processing unit 13 can detect the positions of microparticles in real time.

[0142] (2) Method for Tracking the Position of Microparticles The information processing unit 13 does not search for a single microparticle in the entire image, but sets the area around the microparticle in a certain frame image as a region of interest (ROI), and searches within the ROI in the next frame image to detect the microparticle. The information processing unit 13 tracks the microparticle by predicting the movement of the microparticle and changing the ROI for each frame.

[0143] There is a possibility that a plurality of microparticles are floating in the upper space 231. When tracking a plurality of microparticles individually, it is possible to prevent errors in tracking the microparticles by limiting the search range of each individual microparticle.

[0144] FIG. 14 is a diagram illustrating a method for tracking the positions of microparticles by the information processing unit 13. In FIG.

[0145] In a first step, the information processing unit 13 acquires a difference image Pi10 that indicates the difference between the base image and the frame image Pi1, as shown in the middle left of Fig. 14. Next, the information processing unit 13 sets a predetermined range in the difference image Pi10 as an initial ROI r0. The initial ROI r0 is set as a range of a predetermined number of pixels that includes the pixel corresponding to the position of the cell discharge outlet 221.

[0146] The speed of the microparticles at the moment they are ejected from the cell ejection port 221 is faster than the speed of the microparticles when they move within the chamber 201, and there is variation in the direction in which the microparticles are ejected, so it is desirable to set the initial ROI to a wider range than the other ROIs.

[0147] However, the initial ROI must be set so that the preceding particle and the following particle are not included in one initial ROI. Therefore, the initial ROI is optimized based on the ejection speed of the microparticles, the sorting interval of the sorting unit 14, and the frame rate of the imaging unit 23. The size (number of pixels) of the initial ROI may be set automatically during advance calibration or may be specified by the user.

[0148] The information processing unit 13 searches for a microparticle P51 that was introduced into the chamber 201 (discharged from the cell discharge port 221) at time t1 within the initial ROI r0 of the difference image Pi10. When a microparticle is detected within the initial ROI r0, tracking of the microparticle begins.

[0149] In the second step, the information processing unit 13 calculates a movement vector indicating the direction and speed of movement from the position of the cell outlet 221 for the microparticle P51 detected within the initial ROI r0, and sets the range in which the microparticle P51 is predicted to exist in the difference image of the next frame as ROI r1.

[0150] To prevent false detection of microparticles, it is desirable to make the ROI as narrow as possible when tracking the target microparticle, but the ROI must be set to a certain extent so as not to lose track of the target microparticle. The ROI is optimized based on the liquid delivery speed from the liquid delivery pump, the interval at which the target microparticles are selected by the sorting unit 14, and the frame rate of the imaging unit 23.

[0151] In a third step, the information processing unit 13 searches for the microparticle P51 at time t2 within ROI r1 of the difference image Pi11. For the microparticle P51 detected in the difference image Pi11, the information processing unit 13 calculates a movement vector indicating the direction and speed of movement from the position of the microparticle P51 detected in the difference image Pi10, and sets the range in the difference image of the next frame in which the microparticle P51 is predicted to exist as ROI r2.

[0152] If the microparticle P51 cannot be detected within the ROI r1 of the difference image Pi11, the information processing unit 13 determines that the microparticle P51 has been lost and stops tracking the position of the microparticle P51.

[0153] In the fourth step, the information processing unit 13 repeatedly performs the third step. In the example of Fig. 14, the information processing unit 13 searches for a microparticle P51 at time t3 within ROI r2 of the difference image Pi12, calculates the movement vector of the microparticle P51, and sets ROI r3. Next, the information processing unit 13 searches for a microparticle P51 at time t4 within ROI r3 of the difference image Pi13, calculates the movement vector of the microparticle P51, and sets ROI r4.

[0154] In a fifth step, if the microparticle P51 cannot be detected within the ROI r4 of the difference image Pi14, the information processing unit 13 determines that the microparticle P51 has been captured in the well 151 and ends tracking of the microparticle P51. Note that since the microparticle P51 may not have been captured but may have been lost by the information processing unit 13, the information processing unit 13 searches for the microparticle P51 at time t5 within the ROI r4 of the difference image P21. If the microparticle P51 cannot be detected within the ROI r4 of the difference image Pi21, the information processing unit 13 determines that the microparticle P51 has been lost.

[0155] If the loss of microparticles occurs frequently when the processes from the first step to the fifth step are performed, the size (number of pixels) of the initial ROI and other ROIs is reset.

[0156] (3) Method for Tracking the Positions of Multiple Microparticles The following describes a method for simultaneously tracking the positions of multiple microparticles by the information processing unit 13. The information processing unit 13 searches for microparticles discharged from the cell discharge port 221 within the initial ROI for each frame, sets an individual ROI for each microparticle detected within the initial ROI, and searches for each microparticle within the ROI, thereby tracking the positions of the multiple microparticles.

[0157] FIG. 15 is a diagram for explaining a method for tracking the positions of microparticles by the information processing unit 13 when multiple microparticles are floating in the chamber 201.

[0158] As shown in the upper part of Fig. 15, it is assumed that frame images Pi51 to Pi55 are captured by the imaging unit 23 at times t11 to t15, respectively. In the example of Fig. 15, a microparticle P61 is introduced into the chamber 201 at time t11, the microparticle P61 moves at times t12 and t13, and the microparticle P61 is captured in the well 151-9 at time t4. Furthermore, a microparticle P62 is introduced into the chamber 201 at time t12, the microparticle P62 moves at times t13 and t14, and the microparticle P62 is captured in the well 151-13 at time t15.

[0159] In the lower part of Fig. 15, difference image Pi60 shows the difference between the base image and frame image Pi51. Difference image Pi61 shows the difference between frame image Pi51 and frame image Pi52, and difference image Pi62 shows the difference between frame image Pi52 and frame image Pi53. Difference image Pi63 shows the difference between frame image Pi53 and frame image Pi54, and difference image Pi64 shows the difference between frame image Pi54 and frame image Pi55.

[0160] First, the information processing unit 13 sets a predetermined range in the difference image Pi60 as an initial ROI r10. The initial ROI r10 is set as a range of a predetermined number of pixels including the pixel corresponding to the position of the cell outlet 221.

[0161] The information processing unit 13 searches for the microparticle P61 introduced into the chamber 201 (discharged from the cell discharge port 221) at time t11 within the initial ROI r10 of the difference image Pi10. When the microparticle P61 is detected within the initial ROI r10, tracking of the microparticle P61 is started.

[0162] Next, the information processing unit 13 calculates a movement vector indicating the direction and speed of movement from the position of the cell outlet 221 for the microparticle P51 detected within the initial ROI r10, and sets the range in which the microparticle P61 is predicted to exist in the difference image of the next frame as ROI r11.

[0163] Next, the information processing unit 13 searches for the microparticle P62 introduced into the chamber 201 (discharged from the cell discharge port 221) at time t12 within the initial ROI r10 of the difference image Pi61. For the microparticle P62 detected within the initial ROI r10, the information processing unit 13 calculates a movement vector indicating the movement direction and movement speed from the position of the cell discharge port 221, and sets the range in the difference image of the next frame in which the microparticle P62 is predicted to exist as ROI r21.

[0164] In parallel with these processes, the information processing unit 13 searches for the microparticle P61 at time t12 within the ROI r11 of the difference image Pi61. For the microparticle P51 detected in the difference image Pi11, the information processing unit 13 calculates a movement vector indicating the direction and speed of movement from the position of the microparticle P51 detected in the difference image Pi60, and sets the range in the difference image of the next frame in which the microparticle P61 is predicted to exist as ROI r12.

[0165] Next, the information processing unit 13 searches for a microparticle P61 at time t13 within ROI r12 of the difference image Pi62, calculates the movement vector of the microparticle P61, and sets ROI r13. In parallel with this process, the information processing unit 13 searches for a microparticle P62 at time t13 within ROI r21 of the difference image Pi62, calculates the movement vector of the microparticle P62, and sets ROI r22.

[0166] Next, the information processing unit 13 searches for a microparticle P61 at time t14 within ROI r13 of the difference image Pi63, calculates the movement vector of the microparticle P61, and sets ROI r14. In parallel with this process, the information processing unit 13 searches for a microparticle P62 at time t14 within ROI r22 of the difference image Pi63, calculates the movement vector of the microparticle P62, and sets ROI r23.

[0167] Next, if the microparticle P61 cannot be detected within the ROI r14 of the difference image Pi64, the information processing unit 13 determines that the microparticle P61 has been captured in the well 151 and ends tracking of the microparticle P61. In parallel with this process, the information processing unit 13 searches for the microparticle P62 at time t15 within the ROI r23 of the difference image Pi64, calculates the movement vector of the microparticle P62, and sets the ROI r24.

[0168] Even when tracking multiple microparticles, it is important to set the initial ROI so that multiple microparticles are not included within the initial ROI in the difference image of the same frame. Even if multiple microparticles are detected within the ROI, it is possible to predict the range in which each microparticle is expected to exist in the difference image of the next frame, so tracking of the position of each microparticle continues.

[0169] When tracking multiple microparticles, the more microparticles floating in chamber 201 at the same time, the higher the possibility of a tracking error occurring or of reaching the limit of the processing capacity of information processing unit 13. Therefore, in order to identify the position of well 151 in which a microparticle is captured with higher precision, it is desirable to control the flow rate of the microparticles in chamber 201 so that Ns≦Nc, as described above.

[0170] On the other hand, in order to improve the speed of index sorting, it is necessary to shorten the sorting interval of the cell sorter 21 and increase the flow velocity of the microparticles in the chamber 201, and to achieve these, it is desirable to have a high frame rate of the imaging unit 23. The shorter the distance traveled by the microparticles between frames, the narrower the ROI can be, making tracking errors less likely to occur.

[0171] The EVS can capture images at a frame rate of 1000 fps or more, and can adequately track the position of each microparticle even if 100 microparticles are introduced into the chamber 201 per second.

[0172] <Information Processing Unit> FIG. 16 is a block diagram showing an example of the functional configuration of the information processing unit 13.

[0173] As shown in FIG. 16, the information processing unit 13 includes an optical data acquisition unit 301 , a sorting control unit 302 , an image acquisition unit 303 , an image analysis unit 304 , and a linking unit 305 .

[0174] The optical data acquisition unit 301 acquires optical data from the detection unit 12 and supplies it to the sorting control unit 302 and the linking unit 305 .

[0175] The sorting control unit 302 controls the sorting unit 14 of the cell sorter 21. Specifically, the sorting control unit 302 recognizes a plurality of microparticles flowing through the flow path C based on the optical data supplied from the optical data acquisition unit 301. When the sorting control unit 302 recognizes a target microparticle, it controls the sorting unit 14 to select the target microparticle as a microparticle to be collected.

[0176] The sorting control unit 302 notifies the image analysis unit 304 and the linking unit 305 of the sorting number of the microparticles that have been sorted by the sorting unit 14 as microparticles to be sorted (introduced into the chamber 201).

[0177] The image acquisition unit 303 acquires image data obtained by the imaging unit 23 capturing an image of the microwell array 22 and the cell discharge port 221 , and supplies the image data to the image analysis unit 304 .

[0178] The image analysis unit 304 analyzes the image data supplied from the image acquisition unit 303. Specifically, the image analysis unit 304 identifies each microparticle in the chamber 201 using the sorting number of the microparticle introduced into the chamber, tracks the position of the microparticle introduced into the upper space 231 in the chamber 201, and specifies the coordinates of the well 151 in which the microparticle is captured. The sorting number can also be said to be identification information for identifying the microparticle introduced into the chamber 201. The image analysis unit 304 supplies information indicating the coordinates of the well 151 in which the microparticle is captured to the linking unit 305 as position information of the captured microparticle.

[0179] The analysis of the image data may be performed in synchronization with the imaging by the imaging unit 23, or may be performed after the sorting of the plurality of microparticles is completed. When the analysis of the image data is performed after the sorting of the plurality of microparticles is completed, the optimization of the ROI size can be repeatedly performed, and therefore the coordinates of the well 151 in which the microparticles are captured can be identified with high accuracy.

[0180] The linking unit 305 links the position information of the captured microparticle supplied from the image analysis unit 304 with the optical data about the microparticle supplied from the optical data acquisition unit 301 using the sorting number of the microparticle, and records them.

[0181] <<2. Operation of Particle Analysis System>> Next, with reference to the flowchart in FIG. 17, the processing performed by the particle analysis system 1 having the above-described configuration will be described.

[0182] In step S1, the information processing unit 13 starts sending liquids to the cell sorter 21. To the cell sorter 21, a sample liquid containing microparticles and a sheath liquid are sent.

[0183] In step S2, the detection unit 12 performs optical detection to detect the intensity of light generated by irradiating the microparticles with light.

[0184] In step S3, the detector 12 performs signal processing on the electrical signal (detection signal) acquired by optical detection to generate optical data. The sorting control unit 302 of the information processing unit 13 recognizes the microparticles flowing in the optical detection region 106 of the cell sorter 21 based on the optical data generated by the detector 12.

[0185] In step S4, the sorting control unit 302 determines whether the recognized microparticle is a target microparticle. The target microparticle is set in advance, for example, by a user, and the sorting control unit 302 stores optical data (gating region) for the target microparticle in advance. If the optical data for the target microparticle matches the optical data generated by the detection unit 12, the sorting control unit 302 determines that the recognized microparticle is a target microparticle.

[0186] If it is determined in step S4 that the recognized microparticle is not the target microparticle, in step S5, the cell sorter 21 discards the microparticle flowing in the optical detection region 106. Thereafter, the process returns to step S2, and subsequent processes are performed.

[0187] On the other hand, if it is determined in step S4 that the recognized microparticle is the target microparticle, the sorting control unit 302 determines in step S6 whether the recognized microparticle is a single particle.

[0188] If it is determined in step S6 that the recognized microparticle is not a single particle, the process proceeds to step S5, and the subsequent processes are performed.

[0189] On the other hand, if it is determined in step S6 that the recognized microparticle is a single particle, the sorting control unit 302 determines in step S7 whether the sorting interval by the cell sorter 21 is equal to or longer than a predetermined waiting time tm.

[0190] For example, if a target microparticle that passed through the optical detection region 106 at time t101 is selected as a microparticle to be sorted, and then the target particle is recognized at time t102, the sorting control unit 302 calculates the time interval between times t101 and t102. If the time interval between times t101 and t102 is equal to or greater than the waiting time tm, the sorting control unit 302 determines that the sorting interval will be equal to or greater than the waiting time tm.

[0191] If it is determined in step S7 that the sorting interval is not equal to or greater than the waiting time tm, the process proceeds to step S5, and the subsequent processes are carried out.

[0192] On the other hand, if it is determined in step S7 that the sorting interval is equal to or longer than the waiting time tm, in step S8, the cell sorter 21 selects the target microparticles that have passed through the optical detection region 106 as microparticles to be sorted. The microparticles sorted by the cell sorter 21 are discharged into the chamber 201.

[0193] After the first particle, which is the first selected microparticle, is discharged into the chamber 201, in step S9-1, the image capturing unit 23 captures an image of the entire well region, particularly the first particle.

[0194] After the nth particle, which is the nth selected microparticle, is discharged into the chamber 201, in step S9-n, the image capturing unit 23 captures an image of the entire well region, particularly the nth particle.

[0195] After all of the first to n-th particles have been captured in the well 151, the information processing unit 13 performs image data analysis processing in step S10. The image data analysis processing analyzes image data obtained by the imaging unit 23 capturing an image of the well region, and position information of the captured microparticles and optical data about the microparticles are linked by the sorting number of the microparticles and recorded. Details of the image data analysis processing will be described later with reference to FIG. 18.

[0196] After the image data analysis process is performed, the process ends.

[0197] Next, the image data analysis process performed in step S10 of FIG. 17 will be described with reference to the flowchart of FIG.

[0198] In step S21, the image analysis unit 304 of the information processing unit 13 receives a designation of the size of the ROI. Here, the size of the initial ROI and other ROIs is designated by, for example, the user.

[0199] After the first particle is discharged from the cell discharge port 221 in the image captured by the imaging unit 23, in step S22-1, the image analysis unit 304 searches for the first particle within the initial ROI.

[0200] In step S23-1, the image analysis unit 304 determines whether the first particle has been detected as a single particle. If multiple microparticles are detected within the initial ROI, the image analysis unit 304 determines that the first particle has not been detected as a single particle.

[0201] If it is determined in step S23-1 that the first particle has not been detected as a single particle, then in step S24-1, the image analysis unit 304 determines that the first particle has been lost.

[0202] On the other hand, if it is determined in step S23-1 that the first particle has been detected as a single particle, the image analysis unit 304 calculates a movement vector for the first particle in step S25-1.

[0203] In step S26-1, the image analysis unit 304 sets the ROI for the first particle in the image of the next frame based on the movement vector for the first particle.

[0204] In step S27-1, the image analysis unit 304 sets the next frame as the current frame, and searches for the first particle within the ROI of the image of the current frame.

[0205] In step S28-1, the image analysis unit 304 determines whether or not a first particle has been detected within the ROI of the image of the current frame.

[0206] If it is determined in step S28-1 that the first particle has been detected, the process returns to step S25-1, and the subsequent steps are performed.

[0207] On the other hand, if it is determined in step S28-1 that the first particle has not been detected, the image analysis unit 304 compares the base image with the image of the current frame to determine whether there is a well 151 in which a difference has occurred.

[0208] If it is determined in step S29-1 that there is no well 151 in which a difference has occurred, the process proceeds to step S24-1, and the image analysis unit 304 determines that the first particle has been lost.

[0209] On the other hand, if it is determined in step S29-1 that there is a well 151 in which a difference has occurred, the image analysis unit 304 determines in step S30-1 that the first particle has been captured in the well 151.

[0210] In step S31-1, the image analysis unit 304 outputs to the linking unit 305 the coordinates of the well 151 in which the first particle is captured.

[0211] After the n-th particle is discharged from the cell discharge port 221 in the image captured by the imaging unit 23, the image analysis unit 304 searches for the n-th particle within the initial ROI in step S22-n.

[0212] In step S23-n, the image analysis unit 304 determines whether the n-th particle has been detected as a single particle. If multiple microparticles are detected within the initial ROI, the image analysis unit 304 determines that the n-th particle has not been detected as a single particle.

[0213] If it is determined in step S23-n that the n-th particle has not been detected as a single particle, then in step S24-n, the image analysis unit 304 determines that the n-th particle has been lost.

[0214] On the other hand, if it is determined in step S23-n that the n-th particle has been detected as a single particle, the image analysis unit 304 calculates a movement vector for the n-th particle in step S25-n.

[0215] In step S26-n, the image analysis unit 304 sets the ROI for the n-th particle in the image of the next frame based on the movement vector for the n-th particle.

[0216] In step S27-n, the image analysis unit 304 sets the next frame as the current frame, and searches for the n-th particle within the ROI of the image of the current frame.

[0217] In step S28-n, the image analysis unit 304 determines whether or not the n-th particle has been detected within the ROI of the image of the current frame.

[0218] If it is determined in step S28-n that the n-th particle has been detected, the process returns to step S25-n, and the subsequent processes are carried out.

[0219] On the other hand, if it is determined in step S28-n that the nth particle has not been detected, the image analysis unit 304 compares the base image with the image of the current frame to determine whether there is a well 151 in which a difference has occurred.

[0220] If it is determined in step S29-n that there is no well 151 in which a difference has occurred, the process proceeds to step S24-n, and the image analysis unit 304 determines that the n-th particle has been lost.

[0221] On the other hand, if it is determined in step S29-n that there is a well 151 in which a difference has occurred, the image analysis unit 304 determines in step S30-n that the n-th particle has been captured in the well 151.

[0222] In step S31-n, the image analysis unit 304 outputs to the linking unit 305 the coordinates of the well 151 in which the n-th particle is captured.

[0223] After the coordinates of the captured wells 151 for all of the first to n-th particles have been output or the particles have been determined to be lost, in step S32, the linking unit 305 links the positional information of the captured microparticles to the sorting numbers of the microparticles and records them. The optical data for the microparticles is also linked to the positional information of the captured microparticles by the sorting numbers and recorded.

[0224] Thereafter, the process returns to step S10 in FIG. 17, and the process in FIG. 17 ends.

[0225] As described above, in the particle analysis system 1 of the present technology, microparticles are introduced by the cell discharge port 221 into the chamber 201 (microwell array 22) where the microparticles are individually separated, the imaging unit 23 images the well region within the chamber 201, and the image analysis unit 304 of the information processing unit 13 analyzes the image obtained by the imaging unit 23 capturing the image of the well region, thereby identifying the position within the chamber 201 from which the microparticles were separated.

[0226] By employing a technique for identifying the positions of captured microparticles based on an image obtained by capturing an image of the well region, it is possible to introduce further microparticles into chamber 201 without waiting for the microparticles to be sorted into each well. Therefore, the particle analysis system 1 of the present technology can perform index sorting of a large number of microparticles within a practical time period.

[0227] Furthermore, in the particle analysis system 1 of the present technology, microparticles are sorted into a microwell array 22 in which approximately 40,000 wells 151 are arranged in a 20 mm square area, making it possible to perform index sorting of a large number of microparticles without replacing the well plates.

[0228] <<3. Modifications>> One possible application is to perform secondary analysis (imaging, drug assay, etc.) on a plurality of index-sorted cells within the microwell array 22, and then perform tertiary analysis outside the microwell array 22 on a portion of the plurality of cells recovered from the microwell array 22. In this case, a method for recovering the target cells from the microwell array 22 is required.

[0229] Therefore, the following three methods are conceivable for recovering target cells from the microwell array 22: (1) A method of recovering all cells after binding barcode oligonucleotides that indicate positional information to the cells; (2) A method of destroying unnecessary cells and then recovering the remaining cells; and (3) A method of selectively recovering only the target cells.

[0230] The following describes each of the three methods in detail.

[0231] (1) A method for recovering all cells after binding barcode oligonucleotides that indicate positional information to the cells When the closed chamber 201 described with reference to Fig. 10 is used, the user can easily recover all cells captured in each well 151 by manipulating the flow path. For example, the user can recover all cells by sending a buffer solution from the liquid supply pump 258 into the lower space 232 to suspend the cells (or carriers) in the upper space 231, and then aspirating the buffer solution in the upper space 231 through the cell sorter connection port 211.

[0232] The user binds barcode oligonucleotides indicating the respective positional information to the cells before suspending them in the upper space 231. This makes it possible to link the results of optical detection in the cell sorter 21, the results of secondary analysis in the microwell array 22, and the results of tertiary analysis by the barcode oligonucleotides bound to the cells.

[0233] The barcode oligonucleotide is composed of a sequence of four types of bases (ATGC), and is 4 for the base length n. ｎThere are several types of barcode oligonucleotides. For example, to bind a unique barcode oligonucleotide to each of 10,000 cells, a sequence of 7 bases is used (4 ７ (=16384) In reality, it is necessary to avoid DNA sequences that exist in nature and to perform error correction, so sequences of about 14 bases in length are used.

[0234] For example, a barcode oligonucleotide is attached to the bottom surface of each well 151 of the microwell array 22, and when a cell is captured in the well 151, the barcode oligonucleotide binds to the cell.

[0235] It is also possible to bind the barcode oligonucleotide to cells by using a method in which the two-dimensional barcode substrate and the microwell array 22 are in close contact with each other, and then the barcode oligonucleotide is separated from the two-dimensional barcode substrate and transferred to the microwell array side using a restriction enzyme or photolinker cleavage.

[0236] To read the base sequence of the barcode oligonucleotide, the poly-A sequence of mRNA eluted by, for example, lysing the cell membrane is first hybridized with the poly-T sequence of the barcode oligonucleotide. Next, stable cDNA is generated by reverse transcription, and after PCR amplification, the genetic sequence of the cell and the base sequence of the barcode oligonucleotide are read by a sequencer.

[0237] (2) A method for destroying unnecessary cells and then recovering the remaining cells. The user can destroy unnecessary cells by focusing a laser on the cells using a high-power laser optical system. For example, the user can identify unnecessary cells based on the results of secondary analysis, destroy the unnecessary cells, and then recover only the remaining cells.

[0238] Since the absorption wavelength range of cells is 300 to 800 nm, for example, a laser with a wavelength range of 300 to 800 nm is used to destroy cells. The laser may be a pulsed laser. In this case, the user can control the damage to cells by adjusting the peak power of the pulsed laser and the number of irradiation pulses.

[0239] When cells are held on a carrier, a substance that absorbs the laser wavelength is added to the carrier in advance. The user can destroy the cells held on the carrier by focusing a laser on the carrier. When cells are held on a carrier, the carrier prevents cell fragments from the destroyed cells from being released outside the well 151, and can also prevent other cells from being contaminated by the cell fragments.

[0240] (3) Methods for Selectively Recovering Only Target Cells (3-1) Mechanical Pickup Method When using the open chamber 201 described with reference to Figure 8, the user can use a fine suction tool such as a glass capillary to pick up only the target cells from the microwell array 22. However, since only about two or three cells can be recovered per minute, in reality, the mechanical pick-up method is used when recovering about 100 cells.

[0241] (3-2) Method of Fixing Cells Within Wells with a Cell-Trapping Substance and Cleavage Using a Photocleavable Linker Before the cells are index-sorted, a cell-trapping substance such as an antibody is placed via a photocleavable linker on the surface (wall and bottom) of each well 151 of the microwell array 22. The cells trapped in the well 151 are bound to the well 151 via the cell-trapping substance, and therefore are unlikely to escape outside the well 151.

[0242] By irradiating a specific well 151 with a laser using a laser optical system, the user can decompose only the photocleavable linker on the surface of that well 151, and float only the cells captured in that well 151 in the upper space 231 of the chamber 201. The user can recover the cells floating in the upper space 231 by operating the flow path in the same way as in method (1).

[0243] The user may cut and collect one cell at a time, or may cut and collect multiple cells at once.

[0244] When decomposing a photocleavable linker with a laser, it is desirable to use a laser in the wavelength range not absorbed by cells (near-infrared region of about 1000 nm) so as not to damage the cells. When using a laser in the visible region, which is the wavelength range absorbed by cells, the user must set the minimum output and exposure time necessary to cleave the photocleavable linker.

[0245] (3-3) Cell Recovery Method Using Vibration Due to Pulse Laser Irradiation FIG. 19 is a diagram illustrating a cell recovery method using vibration due to pulse laser irradiation.

[0246] In FIG. 19, the chamber 201 is placed on a slide glass 401, and the chamber 201 is irradiated with observation light L1 from the upper space 231 side of the microscope.

[0247] 19 , a near-infrared light absorbing layer 402 is formed on the surface of the microwell array 22 facing the through-holes 152. The near-infrared light absorbing layer 402 is composed of, for example, noble metal nanoparticles such as platinum or palladium, a dye, or carbon nanotubes. Note that the near-infrared light absorbing layer 402 may also be formed by adding the noble metal nanoparticles, dye, or carbon nanotubes to the well substrate.

[0248] 19, when a near-infrared pulsed laser L2 emitted from a laser optical system is focused by an objective lens 411 from the lower space 232 side and irradiated onto, for example, well 151-4, a thermoelastic wave is generated on the bottom surface of well 151-4, and only the cells captured in well 151-4 fly out of well 151-4. The user can recover the cells floating in the upper space 231 by manipulating the flow path in the same way as in method (1).

[0249] When generating vibrations by irradiating a pulsed laser, it is desirable to use a pulsed laser in the near-infrared wavelength range of 900 to 1400 nm, which is less likely to damage cells. For example, when a λ = 1064 nm Nd-YAG laser was focused onto a spot approximately 4 μm in diameter using an objective lens with an NA = 0.26, it was possible to recover only the desired cells from a PDMS microwell array 22 under the following conditions: pulse width = 1 nsec, pulse frequency = 1 kHz, pulse energy = 5 μJ, and number of irradiation pulses = 2.

[0250] When carriers are captured in each well 151, by placing a near-infrared light absorber on the surface or inside of the carrier, it becomes possible to recover the carriers captured in the well 151 by irradiating it with a pulsed laser without forming a near-infrared light absorption layer 402 on the microwell array 22.

[0251] <Example of Computer Configuration> The above-described series of processes can be executed by hardware or software. When the series of processes is executed by software, the program constituting the software is installed from a program recording medium into a computer incorporated in dedicated hardware, or into a general-purpose personal computer, etc.

[0252] FIG. 20 is a block diagram showing an example of the hardware configuration of a computer that executes the above-described series of processes by a program.

[0253] A CPU (Central Processing Unit) 501 , a ROM (Read Only Memory) 502 , and a RAM (Random Access Memory) 503 are interconnected by a bus 504 .

[0254] An input / output interface 505 is also connected to the bus 504. An input unit 506 including a keyboard, a mouse, etc., and an output unit 507 including a display, a speaker, etc. are connected to the input / output interface 505. Also connected to the input / output interface 505 are a storage unit 508 including a hard disk, a nonvolatile memory, etc., a communication unit 509 including a network interface, etc., and a drive 510 that drives removable media 511.

[0255] In a computer configured as described above, the CPU 501 performs the above-described series of processes by, for example, loading a program stored in the storage unit 508 into the RAM 503 via the input / output interface 505 and the bus 504 and executing it.

[0256] The program executed by the CPU 501 is installed in the storage unit 508 by being recorded on, for example, a removable medium 511 or provided via a wired or wireless transmission medium such as a local area network, the Internet, or digital broadcasting.

[0257] The program executed by the computer may be a program that processes in chronological order according to the order described in this specification, or may be a program that processes in parallel or at the required timing, such as when called.

[0258] In this specification, a system refers to a collection of multiple components (devices, modules (components), etc.), regardless of whether all of the components are housed in the same housing. Therefore, multiple devices housed in separate housings and connected via a network, and a single device housed in a single housing with multiple modules, are both systems.

[0259] The effects described in this specification are merely examples and are not limiting, and other effects may also be present.

[0260] The embodiments of the present technology are not limited to the above-described embodiments, and various modifications are possible without departing from the spirit of the present technology.

[0261] For example, the present technology can be configured as a cloud computing system in which a single function is shared and processed collaboratively by a plurality of devices via a network.

[0262] Furthermore, each step described in the above flowchart can be executed by one device, or can be shared and executed by a plurality of devices.

[0263] Furthermore, when one step includes multiple processes, the multiple processes included in that one step can be executed by one device or can be shared and executed by multiple devices.

[0264] <Examples of Combinations of Configurations> The present technology can also have the following configurations.

[0265] (1) An analytical system comprising: a particle introduction unit that introduces microparticles into a sorting container from which the microparticles are individually sorted; an imaging unit that images the sorting container; and an analysis unit that analyzes the image of the sorting container obtained by the imaging unit to identify the position in the sorting container from which the microparticles have been sorted. (2) The analytical system described in (1), further comprising a sorting unit that selects microparticles to be sorted from a plurality of the microparticles flowing in a flow path, wherein the particle introduction unit introduces the microparticles to be sorted, selected by the sorting unit, into the sorting container. (3) The analytical system described in (2), wherein the analysis unit identifies the microparticles introduced into the sorting container by identification information that indicates the order in which the microparticles to be sorted were selected by the sorting unit. (4) The analysis system according to (3), further comprising a detection unit that irradiates light onto the microparticles flowing in the flow path of the sorting unit and performs optical detection for each microparticle to detect the intensity of light emitted from the microparticles, wherein the sorting unit selects the microparticles to be sorted based on the results of the optical detection by the detection unit. (5) The analysis system according to (4), further comprising a linking unit that links, by the identification information, the positions in the sorting container from which the microparticles to be sorted, identified by the analysis unit, with the results of the optical detection by the detection unit for the microparticles to be sorted. (6) The analysis system according to (4) or (5), wherein, when the target microparticle is recognized based on the results of the optical detection by the detection unit, the sorting unit selects the target microparticle as the microparticle to be sorted. (7) The analysis system according to (6), wherein the sorting unit, when a subsequent target microparticle is recognized after a predetermined waiting time has elapsed since sorting the microparticle to be sorted, sorts the subsequent microparticle as the microparticle to be sorted. (8) The analysis system according to any of (2) to (7), wherein the sorting unit has the flow path through which the selected microparticles to be sorted flow, the flow path being in communication with the particle introduction unit.(9) The analysis system according to any one of (1) to (8), wherein the particle introduction unit introduces a subsequent microparticle into the sorting container before the microparticle introduced previously into the sorting container is sorted. (10) The analysis system according to (9), wherein the analysis unit sets an individual ROI for each microparticle in the image and searches for the microparticle within the ROI, thereby tracking the position of the microparticle from when it is introduced into the sorting container by the particle introduction unit until it is sorted. (11) The analysis system according to any one of (1) to (10), wherein the microparticle is at least one of a cell, a non-cellular microparticle, and a carrier. (12) The analysis system according to any one of (1) to (11), wherein the sorting container is configured with an array of multiple wells that capture and sort the microparticles, and wherein a suction unit is formed on the bottom of each well that suctions the microparticles introduced into the sorting container. (13) The analysis system according to any one of (1) to (12), wherein the imaging unit is configured with an EVS that detects events based on changes in brightness of incident light. (14) The analysis system according to any one of (1) to (12), wherein the imaging unit is configured with a frame-type image sensor that scans all pixels at a predetermined frame rate. (15) The analysis system according to (14), wherein the analysis unit identifies the position in the collection container where the microparticles have been sorted based on the difference between frames of the images captured by the imaging unit. (16) The analysis system according to any one of (1) to (12), (14), and (15), wherein the analysis unit identifies the position in the collection container where the microparticles have been sorted based on the difference between the image obtained by the imaging unit capturing the image of the collection container after the microparticles have been introduced and the image obtained by the imaging unit capturing the image of the collection container before the microparticles have been introduced. (17) The analysis system according to any one of (1) to (16), wherein the analysis unit identifies positions in the collection container from which the microparticles have been collected after the collection of the plurality of microparticles in the collection container has been completed.(18) The analysis system according to any one of (1) to (17), wherein the imaging range of the imaging unit includes the particle introduction unit and the sorting container. (19) An analysis method comprising: introducing microparticles into sorting containers that individually collect the microparticles, imaging the sorting containers, and identifying positions in the sorting containers from which the microparticles have been sorted. (20) A program for causing a computer to execute processes of: introducing the microparticles into sorting containers that individually collect the microparticles, analyzing images obtained by imaging the sorting containers, and identifying positions in the sorting containers from which the microparticles have been sorted.

[0266] 1 particle analysis system, 11 light irradiation unit, 12 detection unit, 13 information processing unit, 14 sorting unit, 21 cell sorter, 22 microwell array, 23 imaging unit, 31 tube, 151 well, 152 through-hole, 201 chamber, 301 optical data acquisition unit, 302 sorting control unit 302, 303 image acquisition unit, 304 image analysis unit, 305 linking unit

Claims

1. An analysis system comprising: a particle introduction unit that introduces microparticles into a collection container from which the microparticles are individually collected; an imaging unit that images the collection container; and an analysis unit that analyzes the image of the collection container obtained by the imaging unit to identify the position within the collection container from which the microparticles were collected.

2. The analysis system according to claim 1, further comprising a sorting section that selects the microparticles to be sorted from the plurality of microparticles flowing within the flow path, and the particle introduction section introduces the microparticles to be sorted, selected by the sorting section, into the sorting container.

3. The analysis system according to claim 2, wherein the analysis unit identifies the microparticles introduced into the sorting container using identification information indicating the order in which the microparticles to be sorted were sorted by the sorting unit.

4. The analysis system according to claim 3, further comprising a detection unit that performs optical detection for each microparticle by irradiating light onto the microparticles flowing within the flow path of the sorting unit and detecting the intensity of light emitted from the microparticles, and the sorting unit selects the microparticles to be separated based on the results of the optical detection by the detection unit.

5. The analysis system according to claim 4, further comprising a linking unit that links the position in the collection container from which the microparticles to be collected, as identified by the analysis unit, to the results of the optical detection by the detection unit for the microparticles to be collected, using the identification information.

6. The analysis system according to claim 4, wherein the sorting unit, when the target microparticle is recognized based on the result of the optical detection by the detection unit, sorts the target microparticle as the microparticle to be separated.

7. The analysis system according to claim 6, wherein when a subsequent target microparticle is recognized after a predetermined waiting time has elapsed since the microparticle to be separated is separated, the sorting unit selects the subsequent microparticle as the microparticle to be separated.

8. The analysis system according to claim 2, wherein the sorting section has a flow path through which the selected microparticles to be sorted flow, the flow path being in communication with the particle introduction section.

9. The analysis system according to claim 1, wherein the particle introduction section introduces the subsequent microparticles into the sorting container before the microparticles introduced previously into the sorting container are sorted.

10. The analysis system according to claim 9, wherein the analysis unit sets an individual ROI for each microparticle in the image and searches for the microparticle within the ROI, thereby tracking the position of the microparticle from the time it is introduced into the sorting container by the particle introduction unit until it is sorted.

11. The analysis system according to claim 1, wherein the microparticles are at least one of cells, non-cellular microparticles, and carriers.

12. The analysis system according to claim 1, wherein the collection container is configured with an array of multiple wells that capture and collect the microparticles, and the bottom of the wells is formed with a suction section that sucks up the microparticles introduced into the collection container.

13. The analysis system according to claim 1, wherein the imaging unit is configured as an EVS that detects events based on changes in the luminance of incident light.

14. The analysis system according to claim 1, wherein the imaging unit is configured with a frame-type image sensor that scans all pixels at a predetermined frame rate.

15. The analysis system according to claim 14, wherein the analysis unit identifies the position in the sorting container from which the microparticles have been sorted based on the difference between frames of the images captured by the imaging unit.

16. The analysis system according to claim 1, wherein the analysis unit identifies the position in the collection container from which the microparticle has been collected based on the difference between the image obtained by the imaging unit capturing the collection container after the microparticle has been introduced and the image obtained by the imaging unit capturing the collection container before the microparticle has been introduced.

17. The analysis system according to claim 1, wherein the analysis unit identifies the position in the collection container from which the microparticles have been collected after the collection of the plurality of microparticles in the collection container has been completed.

18. The analysis system according to claim 1, wherein the imaging range of the imaging unit includes the particle introduction unit and the sorting container.

19. An analytical method comprising: introducing microparticles into a collection container from which the microparticles are individually collected; capturing an image of the collection container; and analyzing the image obtained by capturing the image of the collection container to identify the position within the collection container from which the microparticles were collected.

20. A program for causing a computer to execute a process of introducing microparticles into a collection container from which the microparticles are individually collected, analyzing an image obtained by capturing an image of the collection container, and identifying the position in the collection container from which the microparticles were collected.

Images