WO2025205054A1 - Analysis system, analysis method, and program - Google Patents

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

This technology relates to an analysis system, analysis method, and program, and in particular to an analysis system, analysis method, and program that enables index sorting of large numbers of cells within a realistic time frame.

Flow cytometry is a method 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.

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.

If 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.

US Patent Application Publication No. 2017/0122861

Droplet-type cell sorters, which isolate cells into droplets and drop them into each well, can select around 1,000 to 10,000 cells per second and separate the target cells into well plates. However, with droplet-type cell sorters, the droplet drop position varies by millimeters, making it difficult to separate cells into well plates with densely arranged wells. Because cell sorting destinations are limited to, for example, 96-well or 384-well plates, the cell sorter's separation capabilities are not fully utilized.

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, index sorting of more than 10,000 cells is not practical.

This technology was developed in light of these circumstances, and makes it possible to perform index sorting of large numbers of cells within a realistic time frame.

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 of the collection container obtained by the imaging unit to identify the position within the collection container from which the microparticles were collected.

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.

A program according to one aspect of this technology causes a computer to carry out a process of introducing microparticles into a collection container that individually collects the microparticles, 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.

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

1 is a diagram illustrating a schematic configuration example of a particle analysis system according to an embodiment of the present technology. FIG. 10 is a diagram illustrating a more specific configuration example of a particle analysis system. FIG. 1 is a diagram illustrating an example of the configuration of a cell sorter. FIG. 10 is a diagram illustrating a configuration example of a branching section. FIG. 1 shows an example of the appearance of a microwell array. 10A and 10B are diagrams illustrating examples of the shape of a through hole. 10A and 10B are diagrams showing examples of the shape of a well when capturing a carrier. FIG. 1 is a diagram showing an example of the configuration of an open-type chamber. 10A and 10B are diagrams showing examples of images obtained by an imaging unit capturing an image of a well region. FIG. 10 is a diagram showing an example of the configuration of a closed chamber. FIG. 10 is a diagram showing an example of a chamber for collecting microparticles. FIG. 10 is a diagram illustrating a process flow in which an information processing unit tracks the positions of microparticles and records the positions of captured microparticles. FIG. 10 is a diagram illustrating a method for detecting microparticles by an information processing unit. 10A and 10B are diagrams illustrating a method for tracking the positions of microparticles by an information processing unit. 10A and 10B are diagrams illustrating a method for tracking the positions of microparticles by an information processing unit when multiple microparticles are floating in a chamber. FIG. 2 is a block diagram illustrating an example of a functional configuration of an information processing unit. 10 is a flowchart illustrating a process performed by the particle analysis system. 18 is a flowchart illustrating details of the image data analysis process performed in step S10 of FIG. 17. FIG. 10 is a diagram illustrating a cell recovery method using vibrations caused by pulsed laser irradiation. FIG. 2 is a block diagram illustrating an example of the hardware configuration of a computer.

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. Modifications

<<1. Configuration of particle analysis system>>
<Example of particle analysis system configuration>
FIG. 1 is a diagram illustrating a schematic configuration example of a particle analysis system according to an embodiment of the present technology.

The particle analysis system 1 shown in Figure 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 a particle analysis system 1 that includes a sorting unit 14 is a cell sorter.

(biological samples)
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 carrier may hold, for example, a biological component (e.g., a cell or a cell-derived component, such as a secretion). Holding a biological component on the carrier includes, for example, a case in which the biological component is captured on the carrier or a case in which 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 as 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, for example, secretion analysis.

(flow path)
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 surrounded 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.

The particle analysis system 1 is configured so that light from the light irradiation unit 11 is irradiated onto the biological sample flowing within the flow path C, particularly onto microparticles within 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 in a jet-in-air flow cytometer.

(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.

(Detection unit)
The detection unit 12 includes at least one photodetector that detects light generated by irradiating the microparticles with light. 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 photodetector 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.

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 diffraction grating, or a wavelength separation unit such as a dichroic mirror or optical filter. The detection optical system is configured, for example, to disperse light generated by irradiating light onto microparticles, and detect the dispersed light using multiple 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, for example, to separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating light onto the microparticles, and detect the separated light using the corresponding photodetector.

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 the 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 handled 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 fluorescence data, for example. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescence (which may include feature quantities such as Area, Height, and Width).

(Information Processing Department)
The information processing unit 13 includes, for example, a processing unit that processes various data (e.g., optical data) and a memory 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 memory unit may be configured to store the acquired optical data. The memory unit may further be configured to store spectral reference data used in the unmixing processing.

If the particle analysis system 1 includes a sorting unit 14 (described below), the information processing unit 13 can determine whether to sort specific microparticles based on the optical data and/or morphological information. The information processing unit 13 can then control the sorting unit 14 based on the results of this determination, allowing the sorting unit 14 to sort the microparticles.

The information processing unit 13 may be configured to be able 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 be able 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.

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

(Sorting Department)
The sorting unit 14 sorts the 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 the 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.

In the particle analysis system 1, the sorting unit 14 selects and individually dispenses the 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.

Droplet-type cell sorters, which isolate microparticles in droplets and drop them into individual wells, can select approximately 1,000 to 10,000 microparticles per second and separate the desired microparticles into well plates. However, with droplet-type cell sorters, the droplet drop position varies by millimeters, making it difficult to separate microparticles into well plates with densely arranged wells. Since the destination for microparticle collection is limited to, for example, 96-well or 384-well plates, the cell sorter's separation capabilities are not fully utilized.

Microchannel cartridge-type cell sorters have higher precision 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.

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. Since there is no need to move the microwell array when sorting microparticles and it takes less than 10 seconds for a single microparticle to be captured in a well, if a cell sorter capable of high-speed sorting of microparticles is used, it is possible to sort more than 40,000 microparticles in a realistic amount of time.

However, because it is not possible to capture 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.

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.

Figure 2 shows a more specific example configuration of the particle analysis system 1.

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

The cell sorter 21 is a microchannel cartridge type cell sorter that can perform optical detection and sorting of microparticles within a substrate such as plastic or glass. The cell sorter 21 is formed with a channel C and a sorting section 14.

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 irradiated microparticles P. The information processing unit 13 recognizes the irradiated microparticles P 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.

The cell extraction port of the sorting unit 14 is connected to the chamber holding 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 (the order in which they were recognized by the information processing unit 13).

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 flow in the sorting section 14 communicates with an introduction section that introduces the microparticles into the chamber.

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 is formed in the bottom of each well of the microwell array 22, functioning as a suction section for sucking microparticles P into each well. Microparticles P introduced into the chamber are sucked in 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.

However, because it is not possible to control which well a microparticle P is captured in, there is no correlation between the 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.

The imaging unit 23 has pixels arranged in, for example, a two-dimensional grid. The imaging unit 23 is composed of a frame-type image sensor that scans all pixels at a predetermined frame rate and outputs image data (also called frame data), or an EVS (Event-based Vision Sensor) in which event pixels that detect events based on changes in the luminance of incident light are arranged in a two-dimensional grid. Note that instead of frame data, the EVS may output event 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).

The imaging unit 23 captures images of the interior of the chamber and supplies the image data obtained to the information processing unit 13.

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.

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 the optical data (such as the results of optical detection) about that microparticle P using the sorting number.

If additional processes such as imaging, culturing, or reagent reaction assays are performed on the microparticles on the microwell array 22, or the addition of barcode oligonucleotides as preprocessing for genetic analysis, the information processing unit 13 records these results in association with the positional information of each captured microparticle P.

The following describes in detail each component of the particle analysis system 1.

<Cell sorter>
Here, we will describe a cell sorter that selects target cells from a sample of multiple microparticles. Generally, cell sorters are widely used that convert individual microparticles into droplets outside the device, impart a constant positive or negative charge to the droplets containing the target cells, deflect them in a strong electric field, and then 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.

In this technology, 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 extraction outlet and the chamber inlet are connected by a tube or the like, the microparticles are handled in a closed space, making it easy to guide the microparticles into the chamber in the order in which they were sorted in the cell sorter 21.

A microchannel cartridge-type cell sorter selects target microparticles (or microparticles to be discarded) recognized by the information processing unit 13 by manipulating the liquid flow path, for example, by using an actuator to push and pull a channel branch or open and close a valve.

Due to instability caused by disturbances in the liquid flow, the sorting speed of microchannel cartridge-type cell sorters is slower than that of droplet-type cell sorters, which use piezoelectric vibration elements to stably form droplets at a high frequency of around 100 kHz and then isolate and handle the individual microparticles contained within the droplets. However, when index sorting is performed, this is not a problem, as it is sufficient to separate (sort) less than 1% of the entire microparticle sample.

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 caused to microparticles in a microchannel cartridge-type cell sorter is less than that caused by a droplet-type cell sorter. Therefore, microchannel cartridge-type cell sorters are suitable for applications such as culturing cells after sorting.

Figure 3 shows an example configuration of the cell sorter 21.

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

In the cell sorter 21, sample liquid containing microparticles is injected from inlet 101. After sheath liquid is injected from inlet 103, it is split into two at 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 channel 105, the sample liquid meets, sandwiched between sheath liquid, 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 less than 20 μm wide.

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 sample label are detected by the detection unit 12.

Figure 4 is a diagram showing an example configuration of the branching section 107. Figure 4A shows a top view of the branching section 107, and Figure 4B shows an oblique view of the branching section 107.

Among the microparticles flowing through the flow channel 105 after being irradiated with light, the target cells flow linearly through the orifice 121, while the other cells pass through the flow channels 108 that branch off to the left and right of the orifice 121 and are discharged as waste liquid from the outlet 110.

As shown in Figure 4B, buffer solution constantly flows through 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 extraction port 111. While the buffer solution flows in the direction of cell extraction port 111, it also flows in the direction of flow channel 105. The buffer solution creates a flow (block flow) in the opposite direction to the direction of movement of the microparticles, so under normal circumstances, the microparticles do not move in the direction of cell capture chamber 109 or cell extraction port 111.

When the target microparticle approaches the vicinity of the orifice 121, the cell capture chamber 109 is pulled up by the piezoelectric element located directly above, and the microparticle near the orifice 121 is drawn into the cell capture chamber 109 at the same time as the block flow.

The width of the orifice 121 can be changed depending on the diameter of the microparticles. The narrower the width of the orifice 121, the faster the sorting speed will be, as the flow rate required to draw in the microparticles can be achieved with less up and down movement of the cell capture chamber 109. 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 around 50 μm, the width of the orifice 121 can be set to 70 μm.

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

Note that the detection unit 12 is not limited to detecting scattered light or fluorescence generated by laser irradiation. For example, the detection unit 12 may also perform electrical detection or cell morphology imaging.

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

As shown in Figure 5, the microwell array 22 is composed of a plurality of wells 151 arranged in an array on a well substrate. A through-hole 152 is formed in the bottom surface of each well 151.

The microwell array 22 is designed to increase the well surface density compared to conventional 96-well plates (well pitch 9.0 mm) and 384-well plates (well pitch 4.5 mm). For example, the microwell array 22 is designed to reduce the wells 151 to the same size as microparticles and narrow the pitch between the wells 151, allowing index sorting of as many cells as possible.

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

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

(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.

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

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 liquid within the chamber. Furthermore, when a microparticle is captured in well 151, the microparticle blocks through-hole 152, blocking the flow of buffer liquid, preventing other microparticles from being guided to well 151 where a microparticle has already been captured.

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

Figure 6 shows examples of the shape of the through-hole 152.

As shown in Figure 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. As shown in Figure 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.

(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.

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

Therefore, it is desirable to form rectangular through-holes 152 with short sides measuring 3 μm and long sides measuring 10 μm, for example, to prevent cells from passing through while still ensuring a sufficient opening area. The total opening area may also be ensured by forming multiple through-holes 152 with a diameter of 3 μm in one well 151.

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

(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.

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.

Instead of forming a through-hole 152 in part of the bottom surface of the well 151, as shown in Figure 7B, a 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.

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, if Dn = 50 μm, then the desired particle blocking effect will be achieved if Ds = 60 μm and Db = 40 μm. If the through-hole depth H is 80 μm, then the taper angle θ is 7°. This type of taper angle can be added using general machining or lithography, making it possible to produce microwell arrays 22 using a simpler process.

Even when cells themselves are captured rather than carriers, a microwell array 22 can be used in which the through-holes 152, which have a tapered shape in cross section, function as wells 151.

(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.

Possible methods for processing well 151 include mechanical processing, laser drilling, photolithography, 3D printing, injection molding, and other transfer processes, or a combination of these methods.

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 needs to be made of a transparent material. Furthermore, when observing the fluorescence of cells or cell secretions within the wells 151, it is desirable to form the well substrate from a material with sufficiently low autofluorescence. For example, the well substrate is made of COP (cycloolefin polymer) or COC (cycloolefin copolymer).

<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.

Figure 8 shows an example configuration of an open-type chamber 201.

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

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.

Furthermore, a reagent injection port 214 formed on the outside of the chamber 201 is connected to a flow channel formed in the flow channel-equipped resin sheet 215, and the outlet of the flow channel is connected to the upper space 231. The reagent injection port 214 is used to adjust the liquid level in the upper space 231, control the flow rate of the microparticles P in the upper space 231, and inject assay reagents that are performed after the microparticles P have been captured.

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 flow-path resin sheet 212 and are ejected from the cell ejection port 221 into the upper space 231, where they move along with the flow of buffer solution in the upper space 231 and are captured in the well 151. The cell ejection port 221 functions as a particle introduction section that introduces microparticles into the upper space 231 of the chamber 201.

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 interior of the chamber 201 via the waste liquid tube 216. As the buffer liquid filling the lower space 232 is discharged through the waste liquid tube, a flow is created 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 rate of the buffer liquid to set the falling speed of the microparticles P to an appropriate speed.

The upper space 231 is a box-shaped space that is open at the top. Because the upper space 231 is open, specific cells can be extracted from the upper side of the chamber 201 using mechanical methods such as a glass capillary. Furthermore, as indicated by the white arrow in Figure 8, the imaging unit 23 can capture images of the well region of the microwell array 22 and the cell discharge port 221 from the upper space 231 side. If the chamber 201 and the microwell array 22 are made of a transparent material, the imaging unit 23 can also capture images of the well region of the microwell array 22 and the cell discharge port 221 from the lower space 232 side.

Figure 9 shows an example of an image obtained by the imaging unit 23 capturing an image of a well region.

As shown in the upper, middle, and lower rows of Figure 9, the imaging unit 23 captures images of Jurkat cells as microparticles P being sucked into the well 151 and finally captured.

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 position of the wells 151 is 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 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 surface of each well 151. The diameter of the Jurkat cells is an average of 10 μm.

In the image in Figure 9, the objective lens has an NA of 0.25 and a shallow focal depth (5 to 10 μm), so the focus is on the Jurkat cells floating in the upper space 231. However, in implementing this technology, it is necessary to clearly capture the process from when the Jurkat cells 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 sufficient focal depth and a field of view (imaging range) that covers the entire well area. It is also desirable to form the cell discharge outlet 221 as close to the microwell array 22 as possible.

Figure 10 is a diagram showing an example of the configuration of a closed-type chamber 201. In Figure 10, the same components as those described above are assigned the same reference numerals. Duplicate explanations will be omitted where appropriate.

As shown in Figure 10, the upper space 231 is sealed, for example, by a glass lid 251. As indicated by the white arrow in Figure 10, the imaging unit 23 can capture images of the well region of the microwell array 22 from the upper space 231 side. It is easier to use the chamber 201 for automating assays using flow path manipulation and for recovering a large number of cells when the upper space 231 is sealed than when it is open.

Outside the chamber 201, a liquid delivery 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 is connected to the lower space 232, and a liquid delivery 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 within the upper space 231.

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 paths in the resin sheet 215 with flow paths 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.

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 arrows in Figure 11, buffer liquid 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.

In this way, various combinations of flow paths and liquid delivery systems are possible depending on the purpose.

Whether the chamber 201 is open or closed, 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.

It is desirable that the cell extraction port 111 of the cell sorter 21 and the cell sorter connection port 211 of the chamber 201 be connected via as short a path as possible using a tube 31 or the like to prevent the order of the microparticles from being changed along the way.

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 inside the tube forms a laminar flow, and the microparticles move in a straight line while maintaining a constant position across the width of the tube. The flow inside the tube is a Hagen-Poiseuille flow, with a central axis-symmetric flow velocity distribution, where the flow velocity is greatest at the center of the tube and zero at the tube wall.

Microparticles flow at the center of the tube at the highest speed, and as they approach the edges, their speed decreases, so under certain conditions, particles may overtake each other within the tube.

The applicant conducted an experiment in which beads representing cells were flowed through a tube. The results of the experiment are presented below.

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 rate of 0.068 m/s), the time it took for the beads to pass through the tube varied by 36%. The shortest passage time for the beads was 8.0 seconds (flow rate of 0.125 m/s), while the longest was 10.9 seconds (0.092 m/s).

This result means that if the time between when the cell sorter 21 selects the microparticle to be sorted and when it selects 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 between each selection, or any target cells recognized by the information processing unit 13 during that time will be discarded without being selected for sorting.

The time difference Δt between the shortest and longest transit times is proportional to the tube length, so if the tube length is set to 50 mm under the above conditions, for example, 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.

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

Furthermore, when 50 μm diameter beads, which are intended to serve as 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 reduces the degree of freedom for the bead's position across the width of the tube.

From another perspective, 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 variation in the flow speed of particles inside the tube and achieve faster index sorting. In particular, when a carrier that hardly deforms is flowed through the tube, overtaking will not occur if Dt < 2D.

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

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

In principle, the time difference Δt will not become 0, so in the particle analysis system 1 of this technology, a waiting time tm is set by adding a margin to the time difference Δt in order to prevent microparticles from being swapped. 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 information processing unit 13 recognizes a target microparticle after the waiting time tm has elapsed since selecting the target microparticles, the cell sorter 21 selects the target microparticle as a microparticle to be sorted. Therefore, even if the information processing unit 13 recognizes a target microparticle during that period, the target microparticle is discarded. The waiting time tm is specified appropriately by the operator depending on the conditions of use of the system.

(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.

Furthermore, if an excessive number of microparticles are floating in the upper space 231, collisions between the microparticles may change their trajectories, potentially causing tracking by the information processing unit 13 to fail.

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.

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.

Nc≧Ns is achieved 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 selected by the cell sorter 21 per second, the microparticles should travel at an average speed Vc=20 mm/s or greater between the cell discharge outlet 221 and each well 151. Also, when the average distance L is 20 mm and 100 cells are sorted by the cell sorter 21 per second, the microparticles should travel at an average speed Vc=2 m/s or greater between the cell discharge outlet 221 and each well 151.

The flow rate of the microparticles in this 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.

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

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

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. This allows the imaging unit 23 to image the well region at low magnification and with a wide field of view, which ultimately makes it possible to increase the number of wells 151 arranged in the microwell array 22.

<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.

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

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

Next, as shown in the third row of Figure 12, when a second microparticle P32 is captured in well 151-8, the information processing unit 13 detects that a microparticle P31 identified by sorting number 2 has been captured in well 151-8 based on image data obtained by the imaging unit 23 capturing an image of the microwell array 22. The information processing unit 13 links the sorting number (number 2) of microparticle P32 with the positional information of well 151-8 in which microparticle P32 was captured and records them.

By repeating this process, as shown in the fourth row of Figure 12, the information processing unit 13 detects that each of the first to ninth microparticles introduced into the chamber 201 has been captured in a well 151, based on image data obtained by the imaging unit 23 capturing an image of the microwell array 22. The information processing unit 13 links and records the sorting number of each microparticle (numbers 1 to 9) with the positional information of the well 151 in which the microparticle was captured.

(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 .

Figure 13 is a diagram explaining the method for detecting microparticles by the information processing unit 13.

As shown in the upper part of Figure 13, frame images Pi1 to Pi5 are captured by the imaging unit 23 at times t1 to t5, respectively. In the example of Figure 13, microparticle P51 is introduced into chamber 201 at time t1, microparticle P51 moves at times t2 and t3, and microparticle P51 is captured in well 151-9 at time t4.

When taking the difference between consecutive frames in images captured by the imaging unit 23, 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 Figure 13, difference image Pi11 shows the difference between frame image Pi1 and frame image Pi2, and 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.

In the difference image in Figure 13, the areas painted black 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 areas painted white indicate pixels with a pixel value of 1.

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 t1 to time t2. 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 t2 to time t3. Based on the pixel values of the difference image Pi13, 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 Pi14, the information processing unit 13 can detect that the microparticle P1 is stationary from time t4 to time t5, i.e., that the microparticle P1 is captured in the well 151 at time t4.

The information processing unit 13 acquires a difference image Pi21 (bottom of Figure 13) that shows the difference between the base image obtained by the imaging unit 23 capturing an image of the microwell array 22 before the microparticles were introduced into the chamber 201, and the frame image Pi5. In the difference image Pi21, only the pixel corresponding to the position of the microparticle P51 captured in 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 was captured, based on the pixel values of the difference image Pi21.

When the imaging unit 23 is configured with an EVS, a differential image showing the difference between successive frames in images captured by a frame-type image sensor is directly acquired by the imaging unit 23. Because the EVS detects only changes in brightness, the volume of image data acquired by the imaging unit 23 is reduced, allowing the information processing unit 13 to detect the position of microparticles in real time.

(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.

There may be multiple microparticles floating within the upper space 231. When tracking multiple microparticles individually, it is possible to prevent tracking errors by limiting the search range for each individual microparticle.

Figure 14 is a diagram explaining a method for tracking the position of microparticles by the information processing unit 13.

In the first step, the information processing unit 13 acquires a difference image Pi10 that shows the difference between the base image and the frame image Pi1, as shown in the middle left of Figure 14. Next, the information processing unit 13 sets a predetermined range in the difference image Pi10 as the 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.

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 as 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.

However, the initial ROI must be set so that leading and trailing particles are not included in a single initial ROI. Therefore, the initial ROI is optimized based on the microparticle discharge speed, 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.

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. If a microparticle is detected within the initial ROI r0, tracking of the microparticle begins.

In the second step, the information processing unit 13 calculates a movement vector indicating the movement direction and movement speed 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.

In order 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.

In the 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.

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.

In the fourth step, the information processing unit 13 repeats the third step. In the example of FIG. 14, the information processing unit 13 searches for the 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 the 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.

In the fifth step, if the microparticle P51 cannot be detected within 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 simply lost sight of it, the information processing unit 13 searches for the microparticle P51 at time t5 within ROI r4 of the difference image P21. If the microparticle P51 cannot be detected within ROI r4 of the difference image Pi21, the information processing unit 13 determines that the microparticle P51 has been lost.

If microparticles are frequently lost when steps 1 through 5 are performed, the size (number of pixels) of the initial ROI and other ROIs will be reset.

(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.

Figure 15 is a diagram illustrating a method for tracking the positions of microparticles by the information processing unit 13 when multiple microparticles are floating in the chamber 201.

As shown in the upper part of Figure 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 Figure 15, microparticle P61 is introduced into chamber 201 at time t11, microparticle P61 moves at times t12 and t13, and microparticle P61 is captured in well 151-9 at time t4. Furthermore, microparticle P62 is introduced into chamber 201 at time t12, microparticle P62 moves at times t13 and t14, and microparticle P62 is captured in well 151-13 at time t15.

In the lower part of Figure 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.

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

The information processing unit 13 searches for the microparticle P61 introduced into the chamber 201 (ejected from the cell ejection port 221) at time t11 within the initial ROI r10 of the difference image Pi10. If the microparticle P61 is detected within the initial ROI r10, tracking of the microparticle P61 begins.

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.

Next, the information processing unit 13 searches within the initial ROI r10 of the difference image Pi61 for the microparticle P62 introduced into the chamber 201 (discharged from the cell discharge port 221) at time t12. For the microparticle P62 detected within the initial ROI r10, the information processing unit 13 calculates a movement vector indicating the direction and speed of movement 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.

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.

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

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

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

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 will continue.

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

On the other hand, to improve the speed of index sorting, it is necessary to shorten the sorting interval of the cell sorter 21 and increase the flow rate of the microparticles within the chamber 201, and to achieve this, it is desirable to have a high frame rate for 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.

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.

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

As shown in FIG. 16, the information processing unit 13 is composed of 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.

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.

The sorting control unit 302 controls the sorting unit 14 of the cell sorter 21. Specifically, the sorting control unit 302 recognizes multiple microparticles flowing through 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.

The sorting control unit 302 notifies the image analysis unit 304 and the linking unit 305 of the sorting number of the microparticles selected by the sorting unit 14 as microparticles to be separated (introduced into the chamber 201).

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

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, then tracks the position of the microparticle introduced into the upper space 231 within the chamber 201 and identifies the coordinates of the well 151 in which the microparticle was captured. The sorting number can also be considered identification information that identifies 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 was captured to the linking unit 305 as positional information of the captured microparticle.

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 multiple microparticles is complete. When the analysis of the image data is performed after the sorting of multiple microparticles is complete, the ROI size can be optimized repeatedly, allowing the coordinates of the well 151 in which the microparticles are captured to be identified with high accuracy.

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

<<2. Operation of the particle analysis system>>
Next, the processing performed by the particle analysis system 1 having the above configuration will be described with reference to the flowchart of FIG.

In step S1, the information processing unit 13 starts sending liquid to the cell sorter 21. Sample liquid and sheath liquid containing microparticles are sent to the cell sorter 21.

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

In step S3, the detection unit 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 microparticles flowing within the optical detection region 106 of the cell sorter 21 based on the optical data generated by the detection unit 12.

In step S4, the sorting control unit 302 determines whether the recognized microparticle is the target microparticle. The target microparticle is set in advance, for example, by the 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 the target microparticle.

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 within the optical detection region 106. Then, processing returns to step S2, and subsequent processing is performed.

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

If it is determined in step S6 that the recognized microparticle is not a single particle, processing proceeds to step S5, and subsequent processing is performed.

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

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.

If it is determined in step S7 that the sorting interval is not equal to or greater than the waiting time tm, processing proceeds to step S5, and subsequent processing is performed.

On the other hand, if it is determined in step S7 that the sorting interval is equal to or greater than the waiting time tm, then 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.

After the first particle, which is the first selected microparticle, is ejected into the chamber 201, in step S9-1, the imaging unit 23 captures an image of the entire well region, and in particular the first particle.

Furthermore, after the nth particle, which is the nth selected microparticle, is ejected into the chamber 201, in step S9-n, the imaging unit 23 images the entire well region, and in particular the nth particle.

After all of the first particle through the nth particle have been captured in the well 151, in step S10, the information processing unit 13 performs image data analysis processing. The image data analysis processing analyzes the image data obtained by the imaging unit 23 capturing an image of the well region, and the positional information of the captured microparticles and the 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 Figure 18.

After the image data analysis process is completed, the process will end.

Next, the image data analysis process performed in step S10 of Figure 17 will be described with reference to the flowchart in Figure 18.

In step S21, the image analysis unit 304 of the information processing unit 13 accepts the specification of the ROI size. Here, the size of the initial ROI and other ROIs is specified, for example, by the user.

After the first particle is ejected from the cell ejection 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.

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.

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.

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.

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.

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.

In step S28-1, the image analysis unit 304 determines whether a first particle has been detected within the ROI of the image of the current frame.

If it is determined in step S28-1 that the first particle has been detected, processing returns to step S25-1, and subsequent processing is performed.

On the other hand, if it is determined in step S28-1 that the first particle was not 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.

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.

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

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

Furthermore, after the nth particle is ejected from the cell ejection port 221 in the image captured by the imaging unit 23, in step S22-n, the image analysis unit 304 searches for the nth particle within the initial ROI.

In step S23-n, the image analysis unit 304 determines whether the nth 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 nth particle has not been detected as a single particle.

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

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

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

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

In step S28-n, the image analysis unit 304 determines whether the nth particle has been detected within the ROI of the image of the current frame.

If it is determined in step S28-n that the nth particle has been detected, processing returns to step S25-n, and subsequent processing is performed.

On the other hand, if it is determined in step S28-n that the nth particle was not 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.

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

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 nth particle has been captured in the well 151.

In step S31-n, the image analysis unit 304 outputs the coordinates of the well 151 in which the nth particle was captured to the linking unit 305.

After the coordinates of the captured well 151 for all particles 1 through n are output or it is determined that a particle has been lost, in step S32, the linking unit 305 links the position information of the captured microparticle with the sorting number of the microparticle and records it. The optical data for the microparticle is also linked with the position information of the captured microparticle with the sorting number and recorded.

Then, processing returns to step S10 in Figure 17, and the processing in Figure 17 ends.

As described above, in the particle analysis system 1 of the present technology, microparticles are introduced into the chamber 201 (microwell array 22) from which the microparticles are individually separated by the cell discharge port 221, 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 well region, thereby identifying the position within the chamber 201 from which the microparticles were separated.

By using a method for identifying the position of captured microparticles based on the image obtained by capturing an image of the well area, it is possible to introduce more microparticles into the chamber 201 without waiting for the microparticles to be sorted into each well. Therefore, the particle analysis system 1 of this technology is able to perform index sorting of a large number of microparticles within a realistic time frame.

Furthermore, in the particle analysis system 1 of this 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 large quantities of microparticles without having to replace well plates.

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

Therefore, the following three methods are conceivable as methods for recovering target cells from the microwell array 22.
(1) A method of recovering all cells after binding barcode oligonucleotides that indicate their location to the cells. (2) A method of recovering the remaining cells after destroying unnecessary cells. (3) A method of selectively recovering only the target cells.

Below, we'll explain each of the three methods in detail.

(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.

Before suspending the cells in the upper space 231, the user binds barcode oligonucleotides that indicate their respective positional information to the cells. 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 using the barcode oligonucleotides bound to the cells.

Barcode oligonucleotides are composed of a sequence of four types of bases (ATGC), and there are ⁴ⁿ types of barcode oligonucleotides for a base length of n. For example, to bind a unique barcode oligonucleotide to each of 10,000 cells, a sequence of 7 bases is used ( ⁴⁷ = 16384). In practice, sequences of around 14 bases are used because it is necessary to avoid DNA sequences that exist in nature and to perform error correction, etc.

For example, a barcode oligonucleotide is attached to the bottom 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.

Alternatively, while the two-dimensional barcode substrate and microwell array 22 are in close contact with each other, the barcode oligonucleotides can be separated from the two-dimensional barcode substrate and transferred to the microwell array using a restriction enzyme or photolinker cleavage, thereby binding the barcode oligonucleotides to cells.

To read the base sequence of a barcode oligonucleotide, the poly-A sequence of mRNA eluted, for example by dissolving the cell membrane, is first hybridized with the poly-T sequence of the barcode oligonucleotide. Next, stable cDNA is generated through a reverse transcription process, and after PCR amplification, the cell's genetic sequence and the base sequence of the barcode oligonucleotide are read using a sequencer.

(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.

Since the absorption wavelength range of cells is 300 to 800 nm, 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.

When cells are held on a carrier, a substance that absorbs the laser wavelength is added to the carrier beforehand. The user can destroy the cells held on the carrier by focusing the 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 cell fragments, etc.

(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.

(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.

By using a laser optical system to irradiate a specific well 151 with a laser, 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).

The user can cut and collect cells one by one, or cut and collect multiple cells at once.

When using a laser to decompose a photocleavable linker, it is desirable to use a laser in a wavelength range not absorbed by cells (near-infrared region of approximately 1000 nm) so as not to damage the cells. If a laser in the visible region, which is the wavelength range absorbed by cells, is used, the user must set the output and exposure time to the minimum required to cleave the photocleavable linker.

(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.

In Figure 19, the chamber 201 is placed on a glass slide 401, and microscope observation light L1 is irradiated onto the chamber 201 from the upper space 231 side.

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

As shown in Figure 19, when a near-infrared pulsed laser L2 emitted from the 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 at the bottom surface of well 151-4, causing only the cells captured in well 151-4 to fly out of well 151-4. The user can recover cells floating in the upper space 231 by operating the flow path in the same way as in method (1).

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 an Nd-YAG laser with λ = 1064 nm was focused onto a spot approximately 4 μm in diameter using an objective lens with NA = 0.26, it was possible to recover only the desired cells from a microwell array 22 made of PDMS under the following conditions: pulse width = 1 nsec, pulse frequency = 1 kHz, pulse energy = 5 μJ, and number of irradiation pulses = 2.

When carriers are captured in each well 151, by disposing 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 them with a pulsed laser, without having to form a near-infrared light absorbing layer 402 on the microwell array 22.

<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 a general-purpose personal computer.

Figure 20 is a block diagram showing an example of the hardware configuration of a computer that executes the above-mentioned series of processes using a program.

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

Further connected to the bus 504 is an input/output interface 505. Connected to the input/output interface 505 are an input unit 506 consisting of a keyboard, mouse, etc., and an output unit 507 consisting of a display, speakers, etc. Also connected to the input/output interface 505 are a storage unit 508 consisting of a hard disk or non-volatile memory, a communication unit 509 consisting of a network interface, etc., and a drive 510 that drives removable media 511.

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 bus 504 and executing it.

The programs executed by the CPU 501 are stored on removable media 511, or are provided via wired or wireless transmission media such as a local area network, the Internet, or digital broadcasting, and are installed in the storage unit 508.

The program executed by the computer may be a program in which processing is performed chronologically in the order described in this specification, or a program in which processing is performed in parallel or at the required timing, such as when called.

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

The effects described in this specification are merely examples and are not limiting, and other effects may also occur.

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

For example, this technology can be configured as a cloud computing system in which a single function is shared and processed collaboratively by multiple devices over a network.

Furthermore, each step described in the above flowchart can be performed by a single device, or can be shared and executed by multiple devices.

Furthermore, if one step includes multiple processes, the multiple processes included in that one step can be executed by one device, or they can be shared and executed by multiple devices.

<Configuration combination example>
The present technology can also be configured as follows.

(1)
a particle introduction unit that introduces the microparticles into a collection container that individually collects the microparticles;
an imaging unit that images the fraction collection container;
an analyzing unit that analyzes the image obtained by the imaging unit capturing an image of the collection container and identifies the position in the collection container from which the microparticles have been collected.
(2)
a sorting unit that selects the microparticles to be sorted from the plurality of microparticles flowing in the flow channel;
The analysis system according to (1), wherein the particle introduction unit introduces the microparticles to be sorted by the sorting unit into the sorting container.
(3)
The analysis system according to (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)
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,
The analysis system according to (3), wherein the sorting unit sorts the microparticles to be sorted based on a result of the optical detection by the detection unit.
(5)
The analysis system according to (4), further comprising a linking unit that links the position in the sorting container from which the microparticles to be sorted, as identified by the analysis unit, with the result of the optical detection by the detection unit for the microparticles to be sorted, using the identification information.
(6)
The analysis system described in (4) or (5), wherein the sorting unit, when a 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 described in (6) above, wherein the sorting unit, when a subsequent target microparticle is recognized after a predetermined waiting time has elapsed since sorting the microparticle to be separated, sorts the subsequent microparticle as the microparticle to be separated.
(8)
The analysis system according to any one of (2) to (7), wherein the sorting unit is the flow path through which the selected microparticles to be sorted flow, and the flow path is connected to the particle introduction unit.
(9)
The particle introduction section introduces the subsequent microparticles into the collection container before the microparticles introduced previously into the collection container are collected. The analysis system described in any one of (1) to (8).
(10)
The analysis system described in (9) above, 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 any one of (1) to (10), wherein the microparticles are at least one of cells, non-cellular microparticles, and carriers.
(12)
the sorting container is configured by arranging a plurality of wells for capturing and sorting the microparticles,
The analysis system according to any one of (1) to (11), wherein a suction portion for suctioning the microparticles introduced into the sorting container is formed on the bottom surface of the well.
(13)
The analysis system according to any one of (1) to (12), wherein the imaging unit is configured by an EVS that detects an event based on a change in luminance 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 sorting container from which the microparticles have been sorted, based on a difference between frames of the image captured by the imaging unit.
(16)
The analysis system described in any one of (1) to (12), (14), and (15), wherein the analysis unit identifies the position in the collection container where the microparticles were collected based on the difference between the image obtained by the imaging unit capturing the collection container after the microparticles were introduced and the image obtained by the imaging unit capturing the collection container before the microparticles were introduced.
(17)
The analysis system according to any one of (1) to (16), 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 any one of (1) to (17), wherein the imaging range of the imaging unit includes the particle introduction unit and the sorting container.
(19)
introducing the microparticles into a collection vessel that individually collects the microparticles;
taking an image of the collection container;
and analyzing an image obtained by capturing an image of the collection container to identify the position in the collection container from which the microparticles were collected.
(20)
On the computer,
introducing the microparticles into a collection vessel that individually collects the microparticles;
A program for executing a process of analyzing an image obtained by capturing an image of the collection container and identifying the position in the collection container from which the microparticles have been collected.

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 (20)

a particle introduction unit that introduces the microparticles into a collection container that individually collects the microparticles;
an imaging unit that images the fraction collection container;
an analyzing unit that analyzes the image obtained by the imaging unit capturing an image of the collection container and identifies the position in the collection container from which the microparticles have been collected. a sorting unit that selects the microparticles to be sorted from the plurality of microparticles flowing in the flow channel;
The analysis system according to claim 1 , wherein the particle introduction unit introduces the microparticles to be sorted, which have been selected by the sorting unit, into the sorting container. 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. a detection unit that irradiates light onto the microparticles flowing through the flow path of the sorting unit and performs optical detection for each microparticle to detect the intensity of light emitted from the microparticles,
The analysis system according to claim 3 , wherein the sorting unit sorts the microparticles to be sorted based on a result of the optical detection by the detection unit. The analysis system according to claim 4, further comprising a linking unit that links, using the identification information, the position in the sorting container from which the microparticles to be sorted, as identified by the analysis unit, with the result of the optical detection by the detection unit for the microparticles to be sorted. The analysis system according to claim 4 , wherein when the target microparticle is recognized based on the result of the optical detection by the detection unit, the sorting unit sorts the target microparticle as the microparticle to be sorted. The analysis system according to claim 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 collected, sorts the subsequent microparticle as the microparticle to be collected. The analysis system according to claim 2 , wherein the sorting unit includes the flow channel through which the selected microparticles to be sorted flow, the flow channel being in communication with the particle introduction unit. The analysis system according to claim 1 , wherein the particle introduction unit introduces the subsequent microparticle into the sorting container before the microparticle introduced previously into the sorting container is sorted. 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. The analysis system according to claim 1 , wherein the microparticles are at least one of cells, non-cellular microparticles, and carriers. the sorting container is configured by arranging a plurality of wells for capturing and sorting the microparticles,
The analysis system according to claim 1 , wherein a suction portion for suctioning the microparticles introduced into the sorting container is formed on the bottom surface of the well. The analysis system according to claim 1 , wherein the imaging unit is configured by an EVS that detects an event based on a change in luminance of incident light. 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. 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 a difference between frames of the images captured by the imaging unit. 2. 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 a 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. The analysis system according to claim 1 , wherein the analysis unit identifies positions in the sorting container from which the microparticles have been sorted after sorting of the plurality of microparticles in the sorting container has been completed. The analysis system according to claim 1 , wherein the imaging range of the imaging unit includes the particle introduction unit and the sorting container. introducing the microparticles into a collection vessel that individually collects the microparticles;
taking an image of the collection container;
and analyzing an image obtained by capturing an image of the collection container to identify the position in the collection container from which the microparticles were collected. On the computer,
introducing the microparticles into a collection vessel that individually collects the microparticles;
A program for executing a process of analyzing an image obtained by capturing an image of the collection container and identifying the position in the collection container from which the microparticles have been collected.