Position adjustment method, microparticle analysis apparatus, and program

Abstract

Provided is a method for adjusting the positional relationship between a flow path position and a light irradiation position.　The present disclosure provides a method for adjusting a position comprising: an imaging step in which, while a flow path through which microparticles can flow is moved in an optical axis direction, said flow path is imaged at a plurality of positions along the optical axis direction; a movement step in which the flow path is moved in the optical axis direction on the basis of a focus index for each of the plurality of images obtained in the imaging step; and an adjustment step in which a characteristic position for the flow path is determined from an image of the flow path in a position after being moved in the movement step, and the positional relationship between the characteristic position and a reference position is adjusted along a direction that is perpendicular to the optical axis direction.

Description

Technical Field

[0001] The present technology relates to a position adjustment method, a microparticle analyzer, and a program. More specifically, it provides a method for adjusting the position of a flow path through which microparticles flow, a microparticle analyzer that executes the position adjustment method, and a program for causing the microparticle analyzer to execute the position adjustment method.

Background Art

[0002] To collect microparticles, various microparticle collection devices have been developed so far. For example, in a particle collection system used in a flow cytometer, a laminar flow composed of a sample liquid containing cells and a sheath liquid is discharged from an orifice formed in a flow cell or a microchip. When discharged, a predetermined vibration is applied to the laminar flow to form droplets. The moving direction of the formed droplets is electrically controlled depending on whether the target particles are included or not, and the target particles are collected.

[0003] Techniques for collecting target particles in a microchip without forming droplets as described above have also been developed. For example, Patent Document 1 below describes "a sample liquid introduction flow path through which a sample liquid containing microparticles flows, at least one pair of sheath liquid introduction flow paths that merge into the sample liquid introduction flow path from both sides thereof and introduce a sheath liquid around the sample liquid, a merging flow path that communicates with the sample liquid introduction flow path and the sheath liquid introduction flow path and through which the liquids flowing through these flow paths merge and flow, a negative pressure suction unit that communicates with the merging flow path and sucks and draws in microparticles to be collected, and at least one pair of waste flow paths provided on both sides of the negative pressure suction unit and communicating with the merging flow path." (Claim 1). In the microchip, the target particles are collected by suction into the negative pressure suction unit.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] In the operation of sorting microparticles, it is possible to irradiate the microparticles flowing in the flow path with light and determine whether to sort the microparticles based on the light generated by the irradiation. In order to perform such determination more appropriately, it is desirable that the light be irradiated at a more appropriate position. Therefore, for example, microparticles are caused to flow in a flow path, and the position of the flow path or the light irradiation position can be adjusted based on the light generated by irradiating the microparticles with light.

[0006] As an example of a document related to such adjustment, the above Patent Document 2 can be cited. The apparatus described in the document uses the detection intensity of light generated by irradiating laser light onto microparticles flowing in a flow path for position adjustment of the flow path.

[0007] However, the microparticles used for such position adjustment are not the objects to be sorted. When the microparticles are rare or expensive cells, it is desirable that the number of cells that are not the objects to be sorted be minimized. Therefore, the present technology aims to reduce the number of microparticles used in the adjustment of the positional relationship between the flow path position and the light irradiation position. [Means for Solving the Problems]

[0008] The present inventors have found that the above problems can be solved by a specific position adjustment method. That is, the present technology includes an imaging step of imaging the flow path at a plurality of positions in the optical axis direction while moving the flow path through which microparticles can flow in the optical axis direction, a moving step of moving the flow path in the optical axis direction based on a focus index for each of the plurality of images obtained in the imaging step, and Identifying a characteristic position of the flow path from an image of the flow path at a position after movement in the movement step, and an adjustment step of adjusting a positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction. Provided is a position adjustment method including this. The focus index may be an index indicating whether the flow path is in focus. The focus index may be a focus index obtained using an autofocus function. The autofocus function may be a function based on image difference. The autofocus function may be a Brenner function. The flow path may be used to irradiate laser light onto microparticles flowing through the flow path and analyze the microparticles based on the light generated by the irradiation. The direction of the optical axis of the laser light may be substantially the same as the optical axis direction in the imaging step. The movement step includes an image identification step of identifying an image that gives a focus index satisfying a predetermined standard from the plurality of acquired focus indices. In the movement step, the flow path may be moved to the position where the image identified in the image identification step was captured. The movement step includes an image identification step of identifying an image that gives a maximum or minimum focus index from the plurality of acquired focus indices. In the movement step, the flow path may be moved to the position where the image identified in the image identification step was captured. The characteristic position may be identified based on the position of the wall defining the flow path. The characteristic position may be a substantially central position in the width direction of the flow path. The reference position may be the position where the optical axis of the laser light irradiated onto the microparticles flowing through the flow path passes. The positional relationship may be a positional relationship in the width direction of the flow path. In the adjustment step, the flow path may be moved in the width direction of the flow path. The flow path may be used to irradiate laser light onto microparticles flowing through the flow path and analyze the microparticles based on the light generated by the irradiation. In the adjustment step, the irradiation position of the laser light can be adjusted. The position adjustment method may further include an additional position adjustment step of irradiating laser light onto microparticles flowing through the flow path and further adjusting the position of the flow path based on the light generated by the irradiation. The microparticles may be biological particles.

[0009] Also, the present technology An imaging optical system that images the flow path at a plurality of positions in the optical axis direction while moving the flow path through which microparticles can flow in the optical axis direction, A movement control unit that moves the flow path in the optical axis direction based on a focus index for each of a plurality of images captured by the imaging optical system, and A position relationship adjustment unit that adjusts the position relationship in a direction perpendicular to the optical axis direction between a feature position specified from an image of the flow path at the moved position and a reference position. A microparticle analysis apparatus including the above is also provided. The microparticle analysis apparatus An irradiation optical system that irradiates laser light onto microparticles flowing through the flow path, and A light detection system that detects the light generated by the irradiation of the laser light May further include the above, The direction of the optical axis of the laser light may be substantially the same as the optical axis direction of the imaging optical system. The microparticle analysis apparatus An irradiation optical system that irradiates laser light onto microparticles flowing through the flow path, and A light detection system that detects the light generated by the irradiation of the laser light May further include the above, One or both of the irradiation optical system and the light detection system may share at least one of the optical components included in the imaging optical system.

[0010] Also, the present technology An imaging step of imaging the flow path through which fine particles can flow while moving the flow path in the optical axis direction at a plurality of positions in the optical axis direction; A moving step of moving the flow path in the optical axis direction based on a focus index for each of the plurality of images obtained in the imaging step, and An adjustment step of specifying a characteristic position of the flow path from an image of the flow path at a position after movement in the moving step and adjusting a positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction A program for causing a fine particle analyzer to execute is also provided.

Brief Description of Drawings

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Embodiments for Carrying Out the Invention

[0012] Hereinafter, preferred embodiments for carrying out the present technology will be described. Note that the embodiments described below show typical embodiments of the present technology, and the scope of the present technology is not limited only to these embodiments. The description of the present technology will be made in the following order. 1. First Embodiment (Position Adjustment Method) (1) Explanation of the First Embodiment (2) An example of a microchip for microparticle separation including a flow path targeted by the position adjustment method of the present technology, and an example of a microparticle separation operation using the microchip for microparticle separation (2-1) Flow-through process (2-2) Judgment process (2-3) Recovery process (2-4) Configuration example of the optical system (2-5) Microchip for microparticle separation and microparticles (3) Processes included in the position adjustment method of the present technology (3-1) Imaging process (3-2) Movement process (3-3) Adjustment process (3-3-1) Feature position (3-3-2) Reference position (3-3-3) Adjustment of the positional relationship (3-3-4) Advantage of performing the movement process before the adjustment process (4) Additional position adjustment process (4-1) First fine adjustment process (4-2) Second fine adjustment process (5) Another example of a microparticle analyzer that executes the position adjustment method of the present technology 2. Second Embodiment (Microparticle Analyzer) 3. Third Embodiment (Program)

[0013] 1. First Embodiment (Position Adjustment Method)

[0014] (1) Description of the First Embodiment

[0015] The position adjustment method of the present technology includes moving a flow path based on the focus index of each of a plurality of flow path images captured at a plurality of different positions in the optical axis direction, and adjusting the positional relationship between a feature position and a reference position specified from the image of the flow path at the position after the movement. By the movement and the adjustment, the flow path can be moved to a preferable position. Further, in the movement and the adjustment, it is not necessary for microparticles to flow in the flow path. Therefore, the microparticles used for position adjustment can be reduced.

[0016] The position adjustment method of the present technology may be performed to adjust the position of a flow path in an apparatus (such as an apparatus for analyzing and / or separating microparticles) where light (such as laser light) is irradiated onto microparticles flowing in the flow path. The flow path may be used, for example, to irradiate laser light onto microparticles flowing through the flow path and perform analysis of the microparticles based on the light generated by the irradiation. The direction of the optical axis of the laser light is preferably substantially the same as the optical axis direction in the imaging step included in the position adjustment method of the present technology. More specifically, the flow path may be, for example, a flow path in a microparticle separation structure (such as a flow cell or a microchip) used in a microparticle analyzer (such as a flow cytometer) that forms droplets to perform analysis and / or separation of microparticles, or a flow path in a microchip used in a microparticle analyzer that performs analysis and / or separation of microparticles in a microchip without forming droplets. The apparatus in which the position adjustment method of the present technology is executed is not limited to these. For example, it may be used in other apparatuses that require adjustment of the positional relationship between the position of the flow path and the irradiation position of light (such as laser light) onto the flow path.

[0017] Hereinafter, first in (2), an apparatus that performs analysis and / or separation of microparticles in a microchip without forming droplets will be described, and then in (3), an example of the execution of the position adjustment method of the present technology in the apparatus will be described.

[0018] (2) Examples of a microparticle separation microchip including a flow path that is the target of the position adjustment method of the present technology, and an example of a microparticle separation operation using the microparticle separation microchip

[0019] FIG. 1 shows a schematic diagram of a configuration example of a microparticle separation microchip including a flow path that is the target of the position adjustment method of the present technology and a configuration example of a microparticle analyzer including the microchip. FIG. 2 shows a configuration example of the optical system of the microparticle analyzer. FIG. 3 shows an example of a flowchart of a microparticle separation operation by the microparticle analyzer.

[0020] The microparticle separation microchip 150 shown in FIG. 1 has a sample liquid flow path 152 and a sheath liquid flow path 154 that joins the sample liquid flow path 152 at a junction 162. The microparticle separation microchip 150 is further provided with a sample liquid inlet 151 and a sheath liquid inlet 153. In FIG. 1, a part of the sheath liquid flow path 154 is shown by a dotted line. The portion shown by the dotted line is at a position lower (shifted in the optical axis direction described later) than the sample liquid flow path 152 shown by a solid line, and at the position where the flow path shown by the dotted line and the flow path shown by the solid line intersect, these flow paths are not in communication. Also, in FIG. 1, the sample liquid flow path 152 is shown as bending twice between the sample liquid inlet 151 and the junction 162, but this is for the purpose of easily distinguishing the sample liquid flow path 152 from the sheath liquid flow path 154. The sample liquid flow path 152 may be linearly configured without bending in this way between the sample liquid inlet 151 and the junction 162. In the microparticle separation operation, a sample liquid containing microparticles is introduced from the sample liquid inlet 151 into the sample liquid flow path 152, and a sheath liquid not containing microparticles is introduced from the sheath liquid inlet 153 into the sheath liquid flow path 154.

[0021] The microparticle separation microchip 150 has a confluent flow path 155 having the junction 162 at one end. The sample liquid and the sheath liquid merge at the merging section 162 and flow through the merging flow path 155 toward the particle separation section 157. In particular, the sample liquid and the sheath liquid merge at the merging section 162 to form a laminar flow in which, for example, the periphery of the sample liquid is surrounded by the sheath liquid. Preferably, fine particles are arranged in a substantially single row in the laminar flow. The flow path structure in which the sample liquid flow path 152 and the two sheath liquid flow paths 154 merge at the merging section 162 and have a merging flow path 155 having the merging section 162 as one end forms a laminar flow containing fine particles flowing in a substantially single row. Thereby, in the light irradiation in the detection region 156 described below, it becomes easier to distinguish the light generated by irradiating one fine particle from the light generated by irradiating other fine particles.

[0022] The microchip 150 for separating fine particles further has a particle separation section 157 at the other end of the merging flow path 155. FIG. 4 shows an enlarged view of the particle separation section 157. As shown in FIG. 4A, at the other end, the merging flow path 155 is connected to the fine particle recovery flow path 159 via the connection flow path 170. As shown in FIG. 4A, the merging flow path 155, the connection flow path 170, and the fine particle recovery flow path 159 may be coaxial. When the particle to be recovered flows into the particle separation section 157, as shown in FIG. 4B, a flow is formed from the merging flow path 155 through the connection flow path 170 into the fine particle recovery flow path 159, and the particle to be recovered is recovered into the fine particle recovery flow path 159. Thus, the particle to be recovered flows through the connection flow path 170 into the fine particle recovery flow path 159. When fine particles that are not the particles to be recovered flow into the particle separation section 157, the fine particles that are not the particles to be recovered flow into the branch flow path 158, as shown in FIG. 4C. In this case, no flow into the fine particle recovery flow path 159 is formed.

[0023] As shown in Fig. 1, the microparticle recovery flow path 159 extends linearly from the particle separation section 157, makes a U-turn, and is formed to reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the microparticle recovery flow path 159 is discharged out of the chip from the recovery flow path end 163. As shown in Fig. 1, the two branch flow paths 158 also extend linearly from the particle separation section 157, make a U-turn, and are formed to reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the branch flow path 158 is discharged out of the chip from the branch flow path end 166. In Fig. 1, for the microparticle recovery flow path 159, the display method is changed to a solid line and a dotted line at the U-turn portion. This change indicates that the position in the optical axis direction changes midway. By changing the position in the optical axis direction in this way, the microparticle recovery flow path 159 and the branch flow path 158 do not communicate at the portion where they intersect. Both the recovery flow path end 163 and the two branch flow path ends 166 are formed on the plane where the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. Further, the introduction flow path inlet 164 for introducing liquid into the introduction flow path 161 is also formed on the said plane. In this way, in the microparticle separation microchip 150, all the inlets where liquid is introduced and the outlets where liquid is discharged are formed on one plane. This facilitates the attachment of the said chip to the microparticle analyzer 100. For example, compared with the case where inlets and / or outlets are formed on two or more planes, the connection between the flow paths provided in the microparticle analyzer 100 and the flow paths of the microparticle separation microchip 150 becomes easier.

[0024] As shown in Figs. 1 and 4, the microparticle separation microchip 150 has an introduction flow path 161 for introducing liquid into the connection flow path 170. By introducing the liquid from the introduction flow path 161 to the connection flow path 170, the inside of the connection flow path 170 is filled with the liquid. Thereby, it is possible to prevent unintended fine particles from entering the fine particle recovery flow path 159.

[0025] The microchip 150 for fine particle separation has two branch flow paths 158 connected to the confluence flow path 155 at the other end of the confluence flow path 155. Thus, in the microchip for fine particle separation used in the present technology, the confluence flow path may branch into the connection flow path and the at least one branch flow path. Fine particles other than the particles to be recovered flow into either of the two branch flow paths 158 without entering the fine particle recovery flow path 159.

[0026] Also, as shown in FIG. 1, the microchip 150 for fine particle separation constitutes a part of the fine particle analyzer 100 including a light irradiation unit 101, a detection unit 102, and a control unit 103 in addition to the microchip.

[0027] Although omitted in FIG. 1, the fine particle analyzer 100 further includes an imaging optical system that images the flow path at a plurality of positions in the optical axis direction while moving the flow path through which the fine particles flow in the optical axis direction. A configuration example of the imaging optical system will be described in detail below with reference to FIG. 2 in “(2-4) Configuration Example of the Optical System”. In “(2-4) Configuration Example of the Optical System” below, the light irradiation unit 101 and the detection unit 102 included in the optical system of the fine particle analyzer 100 are also described.

[0028] The control unit 103 of the fine particle analyzer 100 may include a signal processing unit 104, a determination unit 105, and a separation control unit 106 as shown in FIG. 5. The control unit 103 further includes a movement control unit 108 and a positional relationship adjustment unit 109.

[0029] As described above, the microparticle separation operation using the microparticle separation microchip 150 includes a flowing step S101 of flowing a liquid containing microparticles through the confluence channel 155, a determination step S102 of determining whether the microparticles flowing through the confluence channel 155 are particles to be collected, and a collection step S103 of collecting the particles to be collected into the microparticle collection channel 159, as shown in FIG. 3. Each step will be described below.

[0030] (2-1) Flowing step

[0031] In the flowing step S101, a sample liquid containing microparticles and a sheath liquid not containing microparticles are introduced into the sample liquid channel 152 and the sheath liquid channel 154 from the sample liquid inlet 151 and the sheath liquid inlet 153, respectively.

[0032] The sample liquid and the sheath liquid merge at the merging portion 162 to form a laminar flow in which, for example, the periphery of the sample liquid is surrounded by the sheath liquid. Preferably, the microparticles are arranged in a substantially single row in the laminar flow. That is, in the flowing step S101, a laminar flow containing microparticles flowing in a substantially single row can be formed.

[0033] In this way, in the flowing step S101, the liquid containing microparticles flows through the confluence channel 155, particularly as a laminar flow. The liquid flows through the confluence channel 155 from the merging portion 162 toward the particle separation portion 157.

[0034] (2-2) Determination step

[0035] In the determination step S102, it is determined whether the microparticles flowing through the confluence channel 155 are particles to be collected. The determination can be performed by the determination unit 105. The determination unit 105 can perform the determination based on the light generated by irradiating the microparticles with the light irradiation unit 101. An example of the determination step S102 will be described in more detail below.

[0036] In the determination step S102, the light irradiation unit 101 irradiates the microparticles flowing through the confluence channel 155 (particularly, the channel in the detection region 156) in the microparticle separation microchip 150 with light (for example, excitation light), and the detection unit 102 detects the light generated by the light irradiation. Based on the characteristics of the light detected by the detection unit 102, the determination unit 105 determines whether the microparticle is a particle to be recovered. For example, the determination unit 105 can perform a determination based on scattered light, a determination based on fluorescence, or a determination based on an image (for example, a dark field image or / and a bright field image, etc.). In the recovery step S103 described later, the control unit 103 controls the flow in the microparticle separation microchip 150, so that the particle to be recovered is recovered into the microparticle recovery channel 159.

[0037] The light irradiation unit 101 irradiates the microparticles flowing through the channel in the microparticle separation microchip 150 with light (for example, excitation light). The light irradiation unit 101 may include a light source that emits light and an objective lens that condenses the excitation light onto the microparticles flowing through the detection region. The light source may be appropriately selected by those skilled in the art according to the purpose of the analysis, and may be, for example, a laser diode, an SHG laser, a solid-state laser, a gas laser, a high-intensity LED, or a halogen lamp, or may be a combination of two or more of these. The light irradiation unit may include other optical elements as necessary in addition to the light source and the objective lens.

[0038] In one embodiment of the present technology, the detection unit 102 detects scattered light and / or fluorescence generated from the microparticles by the light irradiation of the light irradiation unit 101. The detection unit 102 may include a condenser lens that condenses the fluorescence and / or scattered light generated from the microparticles and a detector. As the detector, a PMT, a photodiode, a CCD, a CMOS, etc. may be used, but not limited thereto. The detection unit 102 may include other optical elements as necessary in addition to the condenser lens and the detector. The detection unit 102 may further include, for example, a spectroscopic unit. Examples of the optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. By the spectroscopic unit, for example, light of a wavelength to be detected can be separated from light of other wavelengths and detected. The detection unit 102 can convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 102 can further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0039] In another embodiment of the present technology, the detection unit 102 may acquire an image generated by the light irradiation of the light irradiation unit 101. The image may be, for example, a dark field image, a bright field image, or both of them. In this embodiment, the light irradiation unit 101 includes, for example, a halogen lamp or a laser, and the detection unit 102 may include a CCD or a CMOS. The detection unit 102 may be, for example, an imaging device in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are laminated. By operating the DSP of the imaging device as a machine learning unit, the imaging device can operate as a so-called AI sensor. The detection unit 102 including the imaging device can determine whether the microparticle is a particle to be collected, for example, based on a learning model. Further, the learning model may be updated in real time while the method according to the present technology is being performed. For example, during the reset of the pixel array unit in the CMOS sensor, during the exposure of the pixel array unit, or during the readout of the pixel signal from each unit pixel of the pixel array unit, the DSP can perform machine learning processing. Examples of the imaging device operating as an AI sensor include, for example, the imaging device described in International Publication No. WO 2018 / 051809.

[0040] The signal processing unit 104 included in the control unit 103 can process the waveform of the digital electrical signal obtained by the detection unit 102 and generate information (data) regarding the characteristics of light used for the determination by the determination unit 105. As the information regarding the characteristics of the light, the signal processing unit 104 can acquire, from the waveform of the digital electrical signal, for example, one, two, or three of the width of the waveform, the height of the waveform, and the area of the waveform. Further, the information regarding the characteristics of the light may include, for example, the time when the light is detected. The processing by the signal processing unit 104 above can be performed particularly in an embodiment in which the scattered light and / or fluorescence is detected.

[0041] The determination unit 105 included in the control unit 103 determines whether the microparticle is a particle to be collected based on the light generated by irradiating the microparticle flowing in the flow path with light. In an embodiment in which the scattered light and / or fluorescence is detected, the waveform of the digital electrical signal obtained by the detection unit 102 is processed by the control unit 103, and based on the information regarding the characteristics of the light generated by the processing, the determination unit 105 determines whether the microparticle is a particle to be collected. For example, in the determination based on the scattered light, the characteristics of the outer shape and / or internal structure of the microparticle may be specified, and based on the characteristics, it may be determined whether the microparticle is a particle to be collected. Further, for example, by performing pretreatment on the microparticle such as a cell in advance, it is also possible to determine whether the microparticle is a particle to be collected based on the same characteristics as those used in flow cytometry. Further, for example, by labeling the microparticle such as a cell with an antibody or a dye (particularly a fluorescent dye), it is also possible to determine whether the microparticle is a particle to be collected based on the characteristics of the surface antigen of the microparticle. In the embodiment in which the image is acquired, the determination unit 105 included in the control unit 103 determines whether the microparticle is a particle to be collected based on the acquired image (for example, a dark-field image, a bright-field image, or both). For example, based on one or a combination of two or more of the form, size, and color of the microparticle (particularly a cell), it can be determined whether the microparticle is a particle to be collected.

[0042] The determination can be made, for example, by whether information regarding the characteristics of the light satisfies a preset criterion. The criterion can be a criterion indicating that the microparticle is a particle to be collected. The criterion may be appropriately set by those skilled in the art and can be a criterion regarding the characteristics of light, such as a criterion used in technical fields such as flow cytometry.

[0043] One light may be irradiated at one position in the detection region 156, or lights may be irradiated at each of a plurality of positions in the detection region 156. For example, the microchip 150 may be configured such that lights are irradiated at each of two different positions in the detection region 156 (that is, there are two positions in the detection region 156 where lights are irradiated). In this case, for example, it can be determined whether the microparticle is a particle to be collected based on the light (such as fluorescence and / or scattered light, etc.) generated by irradiating the microparticle with light at one position. Further, based on the difference between the detection time of the light generated by the light irradiation at the one position and the detection time of the light generated by the light irradiation at another position, the velocity of the microparticle in the flow path can also be calculated. For this calculation, the distance between the two irradiation positions may be determined in advance, and the velocity of the microparticle can be determined based on the difference between the two detection times and the distance. Further, based on the velocity, the arrival time at the particle sorting unit 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of forming the flow into the microparticle recovery flow path 159 can be optimized. Also, when the difference between the arrival time of a certain microparticle at the particle sorting unit 157 and the arrival time of the microparticle before or after the certain microparticle at the particle sorting unit 157 is equal to or less than a predetermined threshold value, it can also be determined not to collect the certain microparticle. When the distance between the certain microparticle and the microparticle before or after it is small, the possibility that the microparticle before or after it is collected together when the certain microparticle is sucked increases. By determining not to collect the certain microparticle when the possibility of being collected together is high, it is possible to prevent the microparticle before or after it from being collected. Thereby, the purity of the target microparticle among the collected microparticles can be increased. Specific examples of the microchip in which lights are irradiated at each of two different positions in the detection region 156 and the device including the microchip are described, for example, in Japanese Patent Application Laid-Open No. 2014-202573.

[0044] Note that the control unit 103 may control the light irradiation by the light irradiation unit 101 and / or the light detection by the detection unit 102. Further, the control unit 103 may control the driving of a pump for supplying fluid into the microchip 150 for microparticle separation. The control unit 103 may be composed of, for example, a hard disk, a CPU, and a memory in which a program and an OS for causing the microparticle analyzer 100 to execute the position adjustment method according to the present technology are stored. For example, the function of the control unit 103 can be realized in a general-purpose computer. The program may be recorded on a recording medium such as a microSD memory card, an SD memory card, or a flash memory. The program recorded on the recording medium is read by a drive (not shown) provided in the microparticle analyzer 100, and the control unit 103 may cause the microparticle analyzer 100 to execute the position adjustment method according to the present technology and the subsequent microparticle separation operation according to the read program.

[0045] (2-3) Recovery step

[0046] In the recovery step S103, the microparticles determined to be particles to be recovered in the determination step S102 are recovered into the microparticle recovery channel 159. The recovery step S103 is performed in the particle separation unit 157 in the microchip 150. In the particle separation unit 157, the laminar flow that has flowed through the confluence channel 155 branches and flows into two branch channels 158. The particle separation unit 157 shown in FIG. 1 has two branch channels 158, but the number of branch channels is not limited to two. For example, one or a plurality (for example, two, three, or four, etc.) of branch channels may be provided in the particle separation unit 157. The branch channels may be configured to branch in a Y shape on one plane as shown in FIG. 1, or may be configured to branch three-dimensionally.

[0047] An enlarged view of the vicinity of the connection channel 170 is shown in FIGS. 6A and 6B. FIG. 6A is a schematic perspective view of the vicinity of the connection channel 170. FIG. 6B is a schematic cross-sectional view in a plane passing through the center lines of the introduction channel 161 and the connection channel 170. The connection channel 170 includes a channel 170a on the detection region 156 side (hereinafter also referred to as the upstream-side connection channel 170a), a channel 170b on the microparticle recovery channel 159 side (hereinafter also referred to as the downstream-side connection channel 170b), and a connection portion 170c between the connection channel 170 and the introduction channel 161. The introduction channel 161 is provided so as to be substantially perpendicular to the axis of the channel of the connection channel 170. In FIGS. 6A and 6B, two introduction channels 161 are provided so as to face each other at a substantially central position of the connection channel 170, but only one introduction channel may be provided.

[0048] The shape and dimensions of the cross-section of the upstream-side connection channel 170a may be the same as those of the downstream-side connection channel 170b. For example, as shown in FIGS. 6A and 6B, both the cross-section of the upstream-side connection channel 170a and the cross-section of the downstream-side connection channel 170b may be substantially circular having the same dimensions. Alternatively, both of these two cross-sections may be rectangular (such as a square or a rectangle) having the same dimensions.

[0049] Liquid is supplied from the two introduction channels 161 to the connection channel 170 as indicated by the arrows in FIG. 6B. The liquid flows from the connection portion 170c to both the upstream-side connection channel 170a and the downstream-side connection channel 170b.

[0050] When the recovery process is not performed, the liquid flows as follows. The liquid that has flowed into the upstream-side connection channel 170a exits from the connection surface with the confluence channel 155 of the connection channel 170 and then flows separately into the two branch channels 158. By the liquid exiting from the connection surface in this way, it is possible to prevent the liquid and microparticles that do not need to be recovered into the microparticle recovery channel 159 from entering the microparticle recovery channel 159 through the connection channel 170. The liquid that has flowed into the downstream connection channel 170b flows into the fine particle collection channel 159. As a result, the inside of the fine particle collection channel 159 is filled with the liquid.

[0051] Even when the recovery process is performed, the liquid can be supplied from the two introduction channels 161 to the connection channel 170. However, due to the pressure fluctuation in the fine particle collection channel 159, particularly by generating a negative pressure in the fine particle collection channel 159, a flow is formed that flows from the confluence channel 155 through the connection channel 170 into the fine particle collection channel 159. That is, a flow is formed that flows from the confluence channel 155 through the upstream connection channel 170a, the connection part 170c, and the downstream connection channel 170b in this order into the fine particle collection channel 159. As a result, the particles to be recovered are collected in the fine particle collection channel 159.

[0052] The shape and / or dimensions of the cross-section of the upstream connection channel 120a may be different from the shape and / or dimensions of the downstream connection channel 120b. Examples of the different dimensions of these two channels are shown in FIGS. 7A and 7B. As shown in FIGS. 7A and 7B, the connection channel 180 includes a channel 180a on the detection region 156 side (hereinafter also referred to as the upstream connection channel 180a), a channel 180b on the fine particle collection channel 159 side (hereinafter also referred to as the downstream connection channel 180b), and a connection part 180c between the connection channel 180 and the introduction channel 161. The cross-sections of both the upstream connection channel 180a and the downstream connection channel 180b have a substantially circular shape, but the diameter of the cross-section of the latter is larger than the diameter of the cross-section of the former. By making the diameter of the cross-section of the latter larger than that of the former, compared with the case where the diameters of both are the same, it is possible to more effectively prevent the particles to be recovered that have already been separated into the fine particle collection channel 159 immediately after the fine particle separation operation due to the negative pressure described above from being discharged through the connection channel 180 into the confluence channel 155. For example, when both the cross-section of the upstream connection channel 180a and the cross-section of the downstream connection channel 180b are rectangular, by making the area of the cross-section of the latter larger than the area of the cross-section of the former, as described above, it is possible to more effectively prevent the already recovered microparticles from being discharged through the connection channel 180 into the confluence channel 155.

[0053] In the recovery step S103, due to the pressure fluctuation in the microparticle recovery channel 159, the particles to be recovered are recovered into the microparticle recovery channel through the connection channel. This recovery may be performed, for example, by generating a negative pressure in the microparticle recovery channel 159 as described above. The negative pressure may be generated, for example, by deforming the wall defining the microparticle recovery channel 159 by an actuator 107 (particularly a piezo actuator) attached outside the microchip 150. Due to the negative pressure, the flow entering the microparticle recovery channel 159 can be formed. In order to generate the negative pressure, for example, the actuator 107 can be attached outside the microchip 150 so that the wall of the microparticle recovery channel 159 can be deformed. By the deformation of the wall, the internal space of the microparticle recovery channel 159 changes, and a negative pressure can be generated. The actuator 107 can be, for example, a piezo actuator. When the particles to be recovered are sucked into the microparticle recovery channel 159, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can also flow into the microparticle recovery channel 159. In this way, the particles to be recovered are separated in the particle separation unit 157 and recovered into the microparticle recovery channel 159.

[0054] In order to prevent microparticles that are not particles to be recovered from entering the microparticle recovery channel 159 through the connection channel 170, the connection channel 170 is provided with an introduction channel 161. Liquid is introduced into the connection channel 170 from the introduction channel 161. By introducing the liquid, the connection channel 170 is filled with the liquid. Furthermore, a part of the liquid forms a flow from the connection channel 170 toward the confluence channel 155, thereby preventing microparticles other than the particles to be recovered from entering the microparticle recovery channel 159. The liquid that forms the flow from the connection channel 170 toward the confluence channel 155 flows through the branch channel 158 in the same manner as the liquid without flowing through the inside of the confluence channel 155 due to the flow of the liquid flowing through the confluence channel 155 into the branch channel 158. Note that the remainder of the liquid introduced into the connection channel 170 flows into the microparticle recovery channel 159. Thereby, the inside of the microparticle recovery channel 159 can be filled with the liquid.

[0055] The flow that has flowed into the branch channel 158 can be discharged to the outside of the microchip at the branch channel end 160. Also, the particles to be recovered that have been recovered into the microparticle recovery channel 159 can be discharged to the outside of the microchip at the recovery channel end 163. A container can be connected to the recovery channel end 163 via a channel such as a tube. The particles to be recovered may be recovered in the container.

[0056] As shown in FIGS. 1 and 4, in the microparticle separation microchip used in the present technology, the confluence channel, the connection channel, and the recovery channel may be arranged linearly. When these three channels are arranged linearly (particularly coaxially), for example, compared with the case where the connection channel and the recovery channel are arranged at an angle with respect to the confluence channel, the recovery process can be performed more efficiently. For example, the suction amount required to guide the particles to be recovered into the connection channel can be reduced. Also, in the microparticle separation microchip, the microparticles are arranged in a substantially single row in the confluence channel and flow toward the connection channel. Therefore, the suction amount in the recovery process can also be reduced.

[0057] As described above, in the microchip for separating microparticles, liquid is supplied from the introduction channel to the connection channel. Thereby, in the connection channel, a flow is formed that flows from the connection position between the introduction channel and the connection channel toward the confluence channel, preventing the liquid flowing through the confluence channel from invading the connection channel, and also preventing microparticles other than the particles to be recovered from flowing through the connection channel into the recovery channel. When performing the recovery step, as described above, for example, due to the negative pressure generated in the recovery channel, the particles to be recovered are recovered into the recovery channel through the connection channel.

[0058] (2-4) Configuration Example of Optical System

[0059] A configuration example of the optical system constituting the light irradiation unit 101 and the detection unit 102 described above will be described with reference to FIG. 2.

[0060] The optical system 350 shown in FIG. 2 includes a laser light generation unit 351 that generates laser light to be irradiated to the detection region 156. The laser light generation unit 351 includes, for example, laser light sources 352-1, 352-2, and 352-3, and also includes mirror groups 353-1, 353-2, and 353-3 that synthesize the laser light emitted from these laser light sources.

[0061] The laser light sources 352-1, 352-2, and 352-3 emit laser light having different wavelengths from each other. The laser light source 352-1 emits laser light having a wavelength of, for example, 550 nm to 800 nm (for example, a wavelength of 637 nm). The mirror 353-1 has optical characteristics for reflecting the laser light. The laser light source 352-2 emits laser light having a wavelength of, for example, 450 nm to 550 nm (for example, a wavelength of 488 nm). The mirror 353-2 has optical characteristics for reflecting the laser light and transmitting the laser light emitted from the laser light source 352-1. The laser light source 352-3 emits laser light having a wavelength of, for example, 380 nm to 450 nm (for example, a wavelength of 405 nm). The mirror 353-3 has optical characteristics of reflecting the laser light and transmitting the two laser lights emitted from the laser light sources 352-1 and 352-2. By arranging the above three laser light sources and three mirrors as shown in FIG. 2, the laser lights irradiated to the fine particles are synthesized.

[0062] The synthesized laser light passes through the mirror 342, is reflected by the mirror 354, passes through the shutter 355, and enters the objective lens 356. The laser light is condensed by the objective lens 356 and reaches the detection region 156 of the microchip 150. The laser light irradiates the fine particles flowing through the detection region 156, generating fluorescence and scattered light. Thus, in the optical system shown in FIG. 2, the laser light generation unit 351, the mirrors 352 and 354, and the objective lens 356 are included as components of the light irradiation unit 101. Thus, the light irradiation unit used in the present technology may include a laser light generation unit, particularly a laser light generation unit that generates laser light synthesized from a plurality of laser lights. Further, the light irradiation unit used in the present technology may include, for example, an objective lens in addition to the laser light generation unit. Thus, the light irradiation unit that irradiates laser light to the fine particles flowing through the flow path is also referred to as an irradiation optical system in this specification.

[0063] The optical system 350 includes a fluorescence detector 357 that detects the fluorescence. The fluorescence enters the objective lens 356 and is condensed by the objective lens 356. The fluorescence condensed by the objective lens 356 passes through the shutter 355, passes through the mirror 354, and is detected by the fluorescence detector 357.

[0064] The optical system 350 includes a scattered light detector 358-3 that detects backward scattered light among the scattered light. The backward scattered light enters the objective lens 356 and is condensed by the objective lens 356. The backward scattered light condensed by the objective lens 356 passes through the shutter 355, is reflected by the mirror 354, further reflected by the mirror 342, and detected by the scattered light detector 358-3. The scattered light detector 358-3 detects light having the same wavelength as the laser light emitted from the laser light source 352-3 (for example, light having a wavelength of 380 nm to 450 nm).

[0065] The optical system 350 also includes scattered light detectors 358-1 and 358-2 that detect forward scattered light among the scattered light. The forward scattered light enters the objective lens 359 and is condensed by the objective lens 359. The forward scattered light condensed by the objective lens 359 passes through the mirror 343 and is separated into light having the same wavelength as the laser light emitted from the laser light source 352-1 (for example, light having a wavelength of 550 nm to 800 nm) and light having the same wavelength as the laser light emitted from the laser light source 352-2 (for example, light having a wavelength of 450 nm to 550 nm) by the mirror 360. The mirror 360 may be, for example, a half mirror and has optical characteristics of reflecting the former light and transmitting the latter light. The former light is reflected by the mirror 361 and detected by the scattered light detector 358-1. The latter light is detected by the scattered light detector 358-2.

[0066] Thus, in the optical system shown in FIG. 2, a fluorescence detector 357 that detects fluorescence generated by irradiation with laser light, scattered light detectors 358-1, 358-2, and 358-3 that detect scattered light generated by the irradiation, a mirror group that transmits or reflects fluorescence and / or scattered light, and objective lenses 356 and 359 are included as components of the detection unit 102. Thus, the detection unit used in the present technology may include a fluorescence detector and / or a scattered light detector. The detection unit used in the present technology may include, in addition to the fluorescence detector and / or the scattered light detector, an objective lens through which fluorescence and / or scattered light passes. Thus, the detection unit that detects light generated by irradiation of laser light onto microparticles flowing through the flow path is also referred to as an optical detection system in this specification.

[0067] The optical system 350 further includes an illumination device 370 and an imaging device 371. The illumination device 370 and the imaging device 371 are included in an imaging optical system that images the flow path in the imaging step. The imaging optical system shown in FIG. 2 is a configuration example of an imaging optical system of an epi-illumination method. The illumination device 370 irradiates illumination light necessary for imaging the flow path (particularly, the confluence flow path 155 within the detection region 156) of the microparticle separation microchip 150. The illumination light emitted from the illumination device 370 is reflected by the mirror 344 and the mirror 343, and passes through the objective lens 359 to reach the microparticle separation microchip 150. The flow path of the microparticle separation microchip illuminated by the illumination light (particularly, the confluence flow path 156 within the detection region 156) is imaged by the imaging device 371 via the objective lens 359. That is, the illumination device 370 and the imaging device 371 are configured to image the flow path via the objective lens 359. Thus, the illumination device 370, the imaging device 371, and the objective lens 359 may constitute a part of an imaging optical system that images the flow path at a plurality of positions in the optical axis direction. The imaging device 371 may include, for example, a CCD or a CMOS. The illumination device 370 may be, for example, an illumination device used in microscopic observation and may be appropriately selected by those skilled in the art.

[0068] In the present technology, the imaging optical system may be configured to image the flow path through an objective lens through which forward scattered light passes. Alternatively, the imaging optical system may be configured to image the flow path through an objective lens through which backward scattered light and / or fluorescence passes. By sharing at least one optical component such as an objective lens between the light detection system and the imaging optical system in this way, the structure of the optical system can be simplified. Also, in the present technology, the illumination light of the imaging optical system may be irradiated onto the flow path through an objective lens through which the laser light irradiated onto the flow path passes. By sharing at least one optical component such as an objective lens between the illumination optical system and the imaging optical system, the structure of the optical system can be simplified. Hereinafter, the sharing of optical components such as objective lenses will be further described with reference to FIG. 2 below.

[0069] In the optical system 350 shown in FIG. 2, the imaging optical system images the flow path through an objective lens 359 through which the forward scattered light detected by the forward scattered light detectors 358-1 and 358-2 passes. That is, the objective lens 359 is shared by the light detection system and the imaging optical system. Thus, in the present technology, the light detection system may share at least one optical component (for example, an objective lens or a mirror for magnifying the flow path and imaging it on the imaging element) included in the imaging optical system. Thereby, the configuration of the optical system can be simplified. The sharing of the objective lens is particularly preferable from the viewpoints of the performance and / or robustness of the microparticle analyzer. For example, when the imaging optical system and the light detection system for detecting the light generated by irradiating light onto the microparticles use different objective lenses, a shift may occur between the signals detected by the imaging optical system and the light detection system due to the shift of one lens. However, such a shift hardly occurs because the objective lens is shared.

[0070] Also, when the imaging optical system is a transmission illumination system, as will be described later, the illumination light for imaging by the imaging optical system passes through the objective lens 356. As described above, the objective lens 356 also passes the synthesized laser light. Thus, in the present technology, the irradiation optical system may share at least one optical component included in the imaging optical system (such as an objective lens through which the laser light irradiated to the microparticles passes). Thereby, the configuration of the optical system can be simplified.

[0071] Also, when the imaging optical system is a transmission illumination system, as will be described later, the illumination light for imaging by the imaging optical system passes through the objective lens 356. As described above, the objective lens 356 also passes the fluorescence and the backscattered light. As described above, the objective lens 356 also passes the synthesized laser light. Thus, in the present technology, the irradiation optical system and the light detection system may share at least one optical component included in the imaging optical system (such as an objective lens through which the laser light irradiated to the microparticles passes). Thereby, the configuration of the optical system can be simplified.

[0072] In the present technology, the optical axis direction of the imaging optical system and the optical axis direction of the irradiation optical system and / or the optical axis direction of the light detection system may be substantially the same. This will be further described below with reference to FIG. 2.

[0073] In the optical system 350 shown in FIG. 2, the optical axis direction of the imaging optical system (particularly, the optical axis direction of the objective lens 359 for imaging by expanding the flow path with the imaging element 371) is substantially the same as the optical axis direction of the laser light irradiated to the flow path. Thus, in the microparticle analysis apparatus of the present technology, the direction of the optical axis of the laser light irradiated to the microparticles may be substantially the same as the optical axis direction of the imaging optical system.

[0074] Also, in the optical system 350 shown in FIG. 2, the optical axis direction of the imaging optical system (particularly, the optical axis direction of the objective lens 359 for magnifying the flow path and imaging by the imaging element 371) is substantially the same as the optical axis direction of the objective lens 356 through which the fluorescence and the backscattered light generated by the laser light incident on the flow path pass. Thus, in the microparticle analyzer of the present technology, the optical axis direction of the objective lens through which the fluorescence and / or scattered light (particularly, the backscattered light) generated by irradiating the microparticles with the laser light passes and the optical axis direction of the imaging optical system (particularly, the optical axis direction of the objective lens for magnifying the flow path in the imaging optical system and imaging by the imaging device) may be substantially the same.

[0075] Also, in the optical system 350 shown in FIG. 2, the optical axis direction of the imaging optical system (particularly, the optical axis direction of the objective lens 359 for magnifying the flow path and imaging by the imaging element 371) is substantially the same as the optical axis direction of the objective lens 359 through which the forward scattered light generated by the laser light incident on the flow path passes. Thus, in the microparticle analyzer of the present technology, the optical axis direction of the objective lens through which the fluorescence and / or scattered light (particularly, the forward scattered light) generated by irradiating the microparticles with the laser light passes and the optical axis direction of the imaging optical system (particularly, the optical axis direction of the objective lens for magnifying the flow path in the imaging optical system and imaging by the imaging device) may be substantially the same.

[0076] When the imaging element 371 images the detection region 156, the shutter 355 may be closed. Thereby, it is possible to prevent the light caused by the mirrors 352 and 354, the fluorescence detector 357, the scattered light detector 358-3, the laser light generation unit 351, etc. from affecting the imaging.

[0077] As shown in FIG. 2, the illumination device 370 and the imaging element 371 may be configured to obtain an image of the flow path, for example, by an epi-illumination method, particularly, a coaxial epi-illumination method. In the present technology, as long as an image of the flow path can be obtained, another illumination method may be used. For example, a transmission illumination method or a side illumination method may be adopted, and particularly, a transmission illumination method may be adopted. When adopting the transmission illumination method, for example, the laser beam synthesized by the laser beam generation unit 351 may be used as the illumination light. In order to use the laser beam as the illumination light, for example, a diffuser for diffusing the laser beam may be provided on the optical path of the illumination light. The diffuser may be arranged, for example, between the mirror 354 and the objective lens 356. The diffuser may be configured to be movable so that it is arranged on the optical path only when imaging is performed by the imaging optical system. Also, when adopting the transmission illumination method, a combination of an illumination light source and an optical fiber connected to the illumination light source may be used. The light emitted from the optical fiber can be used as the illumination light. The optical fiber can be arranged, for example, so that the light emitted from the optical fiber reaches the microchip 150 for microparticle separation through at least a part of the optical path of the fluorescence detected by the fluorescence detector 357.

[0078] The microchip 150 for microparticle separation can be attached to a stage 374 that can move the microchip in the optical axis direction of the objective lens 359. The stage 374 can be controlled, for example, by the control unit 103 to move in its optical axis direction. In this way, the stage 374 may form a part of the imaging optical system.

[0079] (2-5) Microchip for Microparticle Separation and Microparticles

[0080] In the present technology, "micro" means that at least a part of the flow path included in the microchip for separating microparticles has dimensions on the order of μm, particularly a cross-sectional dimension on the order of μm. That is, in the present technology, a "microchip" refers to a chip including a flow path on the order of μm, particularly a chip including a flow path having a cross-sectional dimension on the order of μm. For example, a chip including a particle separation section composed of a flow path having a cross-sectional dimension on the order of μm can be called a microchip according to the present technology. For example, among the particle separation section 157, the cross-section of the confluence flow path 155 is, for example, rectangular, and the width of the confluence flow path 155 is, for example, 100 μm to 500 μm, particularly 100 μm to 300 μm, within the particle separation section 157. The width of the branch flow path branching from the confluence flow path 155 may be smaller than the width of the confluence flow path 155. The cross-section of the connection flow path 170 is, for example, circular, and the diameter of the connection flow path 170 at the connection portion between the connection flow path 170 and the confluence flow path 155 can be, for example, 10 μm to 60 μm, particularly 20 μm to 50 μm. These dimensions regarding the flow path may be appropriately changed according to the size of the microparticles, particularly the size of the particles to be collected.

[0081] The microchip 150 for separating microparticles can be manufactured by a method known in the art. For example, the microchip 150 for separating biological particles can be manufactured by bonding two or more substrates in which predetermined flow paths are formed. The flow paths may be formed in all of the two or more substrates (particularly two substrates), or may be formed only in a part of the two or more substrates (particularly one of the two substrates). In order to make it easier to adjust the position when bonding the substrates, it is preferable that the flow paths are formed only in one substrate. For example, as shown by the dotted line and the solid line in FIG. 1, a flow path structure in which two flow paths are provided at different positions in the optical axis direction (so as not to communicate with each other) and intersect when viewed from the optical axis direction can be created by laminating three or more substrates in which the flow paths are provided.

[0082] As a material for forming the microparticle separation microchip 150, materials known in the art can be used. For example, polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicon can be mentioned, but are not limited thereto. In particular, since it has excellent processability and can manufacture a microchip inexpensively using a molding device, polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferable.

[0083] The microparticle separation microchip 150 is preferably transparent. For example, in the microparticle separation microchip 150, at least a portion through which light (laser light and scattered light) passes is transparent. For example, the detection region may be transparent. The entire microparticle separation microchip 150 may be transparent.

[0084] In the above, the embodiment in which the above-described flow path group is formed in the disposable microparticle separation microchip 150 has been described. However, in the present technology, the above-described flow path group may not be formed in the microchip 150. For example, the above-described flow path group may be formed in a substrate such as plastic or glass. Further, the above-described flow path group may have a two-dimensional or three-dimensional structure.

[0085] In the present technology, the microparticles may be particles having dimensions that can flow in the flow path in the microparticle separation microchip. In the present technology, the microparticles may be appropriately selected by those skilled in the art. In the present technology, the microparticles may include biological microparticles such as cells, cell aggregates, microorganisms, and liposomes, and synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also referred to as biological particles) can include chromosomes, liposomes, mitochondria, and organelles (cell organelles) that make up various cells. Cells can include animal cells (such as blood cells) and plant cells. Cells can be, in particular, blood cells or tissue cells. The blood cells can be, for example, floating cells such as T cells and B cells. The tissue cells can be, for example, adherent cultured cells or adherent cells separated from tissues. Cell aggregates can include, for example, spheroids and organoids. Microorganisms can include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biological microparticles can also include biological macromolecules such as nucleic acids, proteins, and their complexes. These biological macromolecules can be, for example, those extracted from cells or those contained in blood samples or other liquid samples. Synthetic microparticles can be, for example, microparticles composed of organic or inorganic polymer materials or metals. Organic polymer materials can include polystyrene, styrene - divinylbenzene, and polymethyl methacrylate. Inorganic polymer materials can include glass, silica, and magnetic materials. Metals can include gold colloids and aluminum. The synthetic microparticles can be, for example, gel particles or beads, and more particularly, gel particles or beads to which one or more combinations selected from oligonucleotides, peptides, proteins, and enzymes are bound. The shape of the microparticles can be spherical or substantially spherical, or non - spherical. The size and mass of the microparticles can be appropriately selected by those skilled in the art according to the size of the flow channel of the microchip. On the other hand, the size of the flow channel of the microchip can also be appropriately selected according to the size and mass of the microparticles. In this technology, chemical or biological labels, such as fluorescent dyes or fluorescent proteins, can be attached to the microparticles as needed. By means of such labels, the detection of the microparticles can be made easier. The label to be attached can be appropriately selected by those skilled in the art. Molecules that specifically react with the microparticles (such as antibodies, aptamers, DNA, or RNA) can bind to the label. According to one embodiment of the present technology, the microparticles can be biological particles, particularly cells.

[0086] (3) Steps included in the position adjustment method of the present technology

[0087] An example of the flowchart of the position adjustment method of the present technology is shown in FIG. 8. As shown in FIG. 8, the position adjustment method of the present technology includes an imaging step S201, a movement step S202, and a positional relationship adjustment step S203. Each step will be described below.

[0088] Note that the flow path targeted by the position adjustment method of the present technology may or may not be filled with liquid (it may be empty). As an example of a scenario where the position adjustment method of the present technology is applied to a flow path filled with liquid, the time of priming treatment of the flow path can be cited. For example, when a microchip for microparticle separation is used for the first time, the chip is subjected to a priming treatment of flowing liquid through the flow path, for example, to prevent air bubbles from remaining in the flow path, and then a microparticle separation operation is performed. The position adjustment method of the present technology may be performed during the priming treatment of the flow path. Thereby, since the priming treatment and the position adjustment are executed simultaneously, the time required for the adjustment treatment before the microparticle separation treatment can be shortened.

[0089] (3-1) Imaging step

[0090] As shown in FIG. 2, the microchip 150 for microparticle separation is attached to the stage 374. The microchip 150 for microparticle separation is attached to the stage 374 so that the flow path portion in the detection region 156 of the confluence flow path 155 of the microchip 150 for microparticle separation, particularly the flow path portion irradiated with the laser light synthesized as described above, can be imaged by the imaging device 371 via the objective lens 359.

[0091] Stage 374 is configured to be movable in the optical axis direction (z-axis direction) of the objective lens 359. The optical axis direction can be, for example, the focus direction of imaging in the imaging process, and can also be the same direction as the direction of the optical axis of the objective lens 359. In FIG. 2, the direction shown as the z direction is the optical axis direction. In the imaging process, the optical axis direction is substantially the same as the direction of the optical axis of the laser beam irradiated on the detection region 156 of the microparticle separation microchip 150. Also, stage 374 is configured to be movable in the width direction (x-axis direction) and the axial direction (y-axis direction) of the confluence channel 155. By moving stage 374, the microparticle separation microchip 150 attached to stage 374 and the channels (for example, confluence channel 155) included in the chip also move.

[0092] After the microparticle separation microchip 150 is attached to stage 374, stage 374 can be returned to the origin. The origin is a position preset with respect to the microparticle analyzer 100. And after stage 374 is moved to the origin, it is moved to the measurement start point in the optical axis direction.

[0093] In the imaging process S201, when stage 374 is moved in the optical axis direction from the measurement start point, the confluence channel 155 also moves. The movement ends when stage 374 reaches the measurement end point. The measurement start point and the measurement end point may be appropriately set by those skilled in the art according to the configuration of the microparticle analyzer 100. The movement may be controlled by the control unit 103, and the control unit 103 can move stage 374 in the optical axis direction.

[0094] In the imaging process S201, during the movement of stage 374 in the optical axis direction from the measurement start point to the measurement end point, the imaging element 371 images the channel portion at a plurality of positions in the optical axis direction.

[0095] The stage 374 can be moved in the optical axis direction by, for example, 1 μm to 300 μm, particularly 1.5 μm to 200 μm, more particularly 2 μm to 150 μm at a time. The flow path portion can be imaged at each position after the movement. That is, the interval between the imaged positions may be within this numerical range.

[0096] The obtained plurality of images can be transmitted to the control unit 103 (particularly the movement control unit 108). The plurality of images may be stored, for example, in a storage unit (not shown) connected to the image sensor 371, and the control unit 103 may acquire the plurality of images stored in the storage unit.

[0097] (3-2) Movement step

[0098] The movement control unit 108 calculates a focus index for each of the plurality of images obtained in the imaging step. As a result, a plurality of focus indices are obtained. In this specification, the focus index may mean an index indicating whether the flow path through which fine particles can flow is in focus.

[0099] In a preferred embodiment of the present technology, the focus index is a focus index obtained using an autofocus function. The movement control unit 108 can obtain a focus index for each of a plurality of images acquired in the imaging process using the autofocus function. The autofocus function is preferably any one of a function based on image difference, a function based on the depth of peaks and valleys in a gray-level image, a function based on image contrast, a function based on a histogram, or a function based on a correlation measure. For example, any one of the functions shown in the following formulas (1) to (13) may be used as the autofocus function, and particularly preferably, any one of the following formulas (1) to (3) may be used as the autofocus function. Details of these formulas are described, for example, in A. Santos et al., Evaluation of autofocus functions in molecular cytogenetic analysis, Journal of Microscopy, Vol. 188, Pt3, December (1997).

[0100] Examples of the function based on the image difference include, for example, functions represented by the following formulas (1) to (4). Particularly preferably,

[0101] Formula (1): A function based on the absolute value of the gradient above a threshold

Equation

[0102] Formula (2): A function based on the squared gradient

Equation

[0103] Equation (3): Brenner function

Number

[0104] Equation (4): Function based on absolute gradient value above threshold

Number

[0105] As an example of the function based on the depth of mountains and valleys in the grey level image, for example, the functions represented by the following Equations (5) to (7) can be cited.

[0106] Equation (5): Function based on image content above threshold

Number

[0107] Equation (6): Thresholded pixel count function

Number

[0108] Equation (7): Image power function [Mathematics]

[0109] As an example of the function based on the image contrast, for example, the functions represented by the following formulas (8) to (9) can be cited.

[0110] Formula (8): Function based on deviation [Mathematics]

[0111] Formula (9): Function based on normalized deviation [Mathematics]

[0112] As an example of the function based on the histogram, for example, the functions represented by the following formulas (10) to (11) can be cited.

[0113] Formula (10): Function based on range [Mathematics]

[0114] (11) Function based on entropy [Mathematics]

[0115] As an example of the function based on the image contrast, for example, the functions represented by the following formulas (12) to (13) can be cited.

[0116] Formula (12): Vollath's F 4 Function [Mathematics]

[0117] Equation (13): Vollath's F 5 function

Number

[0118] Particularly preferably, the autofocus function is a function based on image difference. A function based on image difference is preferable from the viewpoint of ease of obtaining a focus index. Also, a function based on image difference is preferable for obtaining a maximum or minimum focus index described later. More preferably, the autofocus function is a Brenner function.

[0119] In the moving step, based on a plurality of acquired focus indices, the flow path is moved in the optical axis direction (for example, the z-axis direction in FIG. 2). For example, the movement may be performed by the movement control unit 108 moving the position of the stage 374. For example, the movement control unit 108 can identify one image based on the focus index and move the flow path to the position where the image was captured. The movement of the flow path in the moving step may be the same as the movement of the flow path in the imaging step, which is a movement by the movement of the stage 374.

[0120] According to one embodiment of the present technology, the moving step may include an image identification step of identifying an image that gives a focus index satisfying a predetermined criterion from the plurality of acquired focus indices. Then, in the moving step, the flow path can be moved to the position where the image identified in the image identification step was captured. The identification of the image can be performed, for example, by the movement control unit 108. As the predetermined criterion, for example, an image that gives a maximum or minimum focus index, or an image that gives a focus index within a predetermined numerical range (particularly, a focus index greater than or equal to a predetermined value or less than or equal to a predetermined value) may be adopted.

[0121] For example, when the value obtained using the autofocus function itself is adopted as the focus index, an image that gives the maximum focus index or an image that gives a focus index equal to or greater than a predetermined value can be identified in the image identification step. An example of a graph plotting a plurality of focus indices against positions in the optical axis direction is shown in FIG. 9. The graph shown in FIG. 9 is a plot of the value itself obtained using the autofocus functions shown in formulas (1) to (3) against the imaging position in the optical axis direction. The value itself is used as the focus index. As shown in FIG. 9, the position where the focus index is maximum can be identified, and the image at that position is identified as the imaging position of the image that gives the maximum focus index.

[0122] Alternatively, when the reciprocal of the value obtained using the autofocus function is adopted as the focus index, an image that gives the minimum focus index or an image that gives a focus index equal to or less than a predetermined value can be identified in the image identification step. An example of a graph plotting a plurality of focus indices against positions in the optical axis direction is shown in FIG. 10. The graph shown in FIG. 10 is a plot of the reciprocal of the value obtained using the autofocus functions shown in formulas (1) to (3) against the imaging position in the optical axis direction. The reciprocal is used as the focus index. As shown in FIG. 10, the position where the focus index is minimum can be identified, and the image at that position is identified as the imaging position of the image that gives the minimum focus index.

[0123] Particularly preferably, the moving step includes an image identification step of identifying an image that gives the maximum or minimum focus index from the plurality of acquired focus indices. Then, in the moving step, the flow path can be moved to the position where the image identified in the image identification step was imaged.

[0124] (3-3) Adjustment step

[0125] The position relation adjustment unit 109 identifies the characteristic position of the flow path from the image of the flow path at the position after movement in the movement step. Then, the position relation adjustment unit 109 adjusts the position relation between the characteristic position and the reference position in a direction perpendicular to the optical axis direction. The position relation may be, for example, a position relation in the width direction and / or the axial direction of the flow path, and particularly, a position relation in the width direction of the flow path.

[0126] (3-3-1) Characteristic position

[0127] The characteristic position is, for example, a position where it is desirable for the microparticles flowing in the flow path to pass through. A more specific example of the characteristic position is a substantially central position in the width direction of the flow path. The width direction of the flow path is a direction perpendicular to the optical axis direction and perpendicular to the axial direction of the flow path (or the flow direction of the microparticles). For example, regarding the flow path P shown on the left in FIG. 11, the x-axis direction corresponds to the width direction. In this figure, the y-axis direction corresponds to the axial direction of the flow path.

[0128] The position relation adjustment unit 109 can identify the characteristic position based on, for example, the position of the wall defining the flow path. The position of the wall can be identified from the image identified in the movement step. The image identified in the movement step is, for example, an image giving the maximum focus index, and is a preferable image from the viewpoint of focus adjustment to the flow path. Therefore, the image identified in the movement step is suitable for accurately identifying the position of the wall defining the flow path.

[0129] The positions of two walls in the width direction of the flow path can be used to identify the characteristic position. For example, when the cross-section of the flow path is circular or rectangular, the shape of the cross-section of the flow path is symmetric with respect to the axis of the flow path. Therefore, the center of the positions of the two walls can be identified as the characteristic position. Thus, in the present technology, the characteristic position may be, for example, a substantially central position in the width direction of the flow path.

[0130] An example of how the position relationship adjustment unit 109 specifies the characteristic position will be described below with reference to FIG. 11. As shown in the left image of FIG. 11, two walls W1 and W2 of the flow path P can be confirmed. The position relationship adjustment unit 109 obtains the pixel value at the position x in the x-axis direction 1 from the position y in the y-axis direction 1 to y 2 across. The position relationship adjustment unit 109 averages the obtained plurality of pixel values to obtain the average pixel value at the position x 1 . The position relationship adjustment unit 109 similarly obtains the average pixel value for other positions in the x-axis direction. The positions in the x-axis direction at which the average pixel values are obtained are set to cross the walls W1 and W2 in the x-axis direction. For example, the average pixel values of each position from x 1 to x 2 shown in FIG. 11 are obtained. x 1 to x 2 When the average pixel values of each position are plotted against the position in the x-axis direction, a graph as shown on the right of FIG. 11 is obtained. Two valleys can be confirmed from the graph. The positions V1 and V2 in the x-axis direction corresponding to the bottoms of these two valleys (the positions in the x-axis direction where the average pixel value is minimized in each valley) correspond to the positions of the walls W1 and W2 in the x-axis direction. The center C of V1 and V2 corresponds to the approximate center position in the width direction of the flow path P. The position relationship adjustment unit 109 can specify the approximate center position as a characteristic position based on the obtained average pixel value. Specifically, the position relationship adjustment unit 109 specifies, for example, the positions in the x-axis direction where the average pixel value is minimized on each side of the axis of the flow path P. The position relationship adjustment unit 109 can specify the center of the two positions in the x-axis direction thus specified as the characteristic position.

[0131] As described above, the positional relationship adjustment unit 109 can specify the characteristic position based on the pixel value of the image of the flow path at the position after movement in the movement step. Preferably, the positional relationship adjustment unit 109 can more specifically specify the characteristic position based on the average pixel value of the image of the flow path (particularly, the average pixel value obtained for each position in the x-axis direction (width direction)). Note that instead of the average pixel value, the pixel values integrated for each position in the x-axis direction (width direction) may be used, or the pixel values at each position in the x-axis direction (width direction) at a certain point in the y-axis direction may be used.

[0132] The image for which the characteristic position is specified may be a grayscale image or a color image. The pixel value may be a pixel value in a grayscale image or a pixel value in a color image. When the image is a color image, the pixel value may be, for example, a pixel value for any one of the R element, G element, and B element.

[0133] (3-3-2) Reference position

[0134] The reference position may be a position that is required to coincide with the characteristic position from the viewpoint of light irradiation on the microparticles. For example, the reference position is a position through which the light irradiated on the microparticles flowing through the flow path passes, and particularly, for example, it is the position through which the optical axis of the laser light irradiated on the microparticles flowing through the flow path passes. The reference position may be determined in advance according to factors such as the structure of the optical system of the microparticle analyzer and the shape of the laser light.

[0135] For example, the reference position may be obtained by preparing a reference microparticle separation microchip, flowing microparticles (such as beads) through the chip, measuring the scattered light and / or fluorescence signals, and adopting the position where these signals are optimal as the reference position. In addition, since the irradiation position of the laser light can be specified by the imaging optical system, the irradiation position (for example, the center position of the laser light) may be adopted as the reference position.

[0136] (3-3-3) Adjustment of positional relationship

[0137] In the adjustment step, the positional relationship adjustment unit 109 adjusts the positional relationship between the feature position and the reference position, and more preferably adjusts the positional relationship in the width direction of the flow path. In one embodiment of the present technology, in the adjustment step, the flow path can be moved in the x-axis direction (width direction) of the flow path. Thereby, the positional relationship in the width direction between the feature position and the reference position can be adjusted. For example, the flow path can be moved in the width direction so that the feature position coincides with the reference position. The positional relationship adjustment unit 109 can drive the movement of, for example, the stage on which the microchip for microparticle separation having the flow path is held, in order to realize the movement of the flow path. In another embodiment of the present technology, in the adjustment step, the irradiation position of the laser light may be adjusted. In this embodiment, the flow path can be used, for example, to irradiate laser light on the microparticles flowing through the flow path and perform analysis of the microparticles based on the light generated by the irradiation. The positional relationship adjustment unit 109 can adjust, for example, the position or orientation of the optical component constituting the optical system for irradiating the laser light, in order to realize the adjustment of the irradiation position.

[0138] (3-3-4) Advantages of performing the movement step before the adjustment step

[0139] Hereinafter, the advantages of performing the movement step before the adjustment step will be described with reference to FIG. 18.

[0140] At the top of FIG. 18, an image obtained by imaging a flow path with an imaging optical system while moving the flow path in the z-axis direction is shown. The z shown at the top of these images is the number of steps of movement. That is, these images are images of the flow path at positions where a certain position on the z-axis is set to 0 and the flow path is moved by 40 steps, 80 steps, 120 steps, 160 steps, 200 steps, and 240 steps, as well as -40 steps, -80 steps, -120 steps, -160 steps, -200 steps, and -240 steps in the z-axis direction. 1 step is approximately 2.5 μm. Among these images, the image at position 0 is the sharpest in focus.

[0141] The plots shown at the bottom of FIG. 18 are plots of the average pixel value with respect to the position in the x-axis direction, created from the images at positions where z is -240 steps, -120 steps, 0, 120 steps, and 240 steps, as described with reference to FIG. 11 at the above-mentioned "(3-3-1) characteristic position". As can be seen from these plots, when z = 0, that is, in the sharpest in focus image, the valley of the average pixel value is the sharpest. Therefore, when z = 0, the position where the average pixel value is the minimum in these valleys is the easiest to identify. Thus, by performing the moving step, it becomes easier to identify the characteristic position in the adjustment step.

[0142] (4) Additional position adjustment step

[0143] The position adjustment method of the present technology may further include an additional position adjustment step of irradiating laser light onto microparticles (such as beads, etc.) flowing through the flow path and further adjusting the position of the flow path based on the light generated by the irradiation. The additional position adjustment step is performed after the adjustment step described in the above "(3-3) adjustment step". By performing the additional position adjustment step, the position of the flow path can be moved to a more appropriate position. An example of a flowchart of the position adjustment method of the present technology including the additional position adjustment step is shown in FIG. 17.

[0144] Among FIGS. 17, the imaging step S201, the moving step S202, and the positional relationship adjustment step S203 are as described in the above (3), so the description thereof is omitted. After the positional relationship adjustment step S203, the additional position adjustment step is performed. The additional position adjustment step may include a first fine adjustment step S300 and a second fine adjustment step S400 as shown in FIG. 17. The first fine adjustment step S300 and the second fine adjustment step S400 correspond to the first fine adjustment step S 22 and the second fine adjustment step S 23 in Japanese Patent Application Laid-Open No. 2016-191715. Therefore, for technical matters related to these steps, please refer to the said publication. Below, the outlines of these steps will be described.

[0145] (4-1) First fine adjustment step FIG. 12 is a diagram for explaining the control in the first fine adjustment step S300. The first fine adjustment step S300 may include, for example, as shown in FIG. 17, a linear signal acquisition step S301, a one-dimensional distribution parameter estimation step S302, and a maximum position movement step S303. Each step will be described.

[0146] (4-1-1) Linear signal acquisition step S301

[0147] In the linear signal acquisition step S301, fluorescence or scattered light is detected from a plurality of detection positions d22 arranged in the width direction (x-axis direction) of the flow path with the feature position (indicated by reference numeral P1 in FIG. 12) after the positional relationship adjustment in the above “(3-3) adjustment step” as the center (FIG. 12A). The detected interval W22 and the number of arrangements of the detection positions d22 can be set as appropriate. In FIG. 12A, the case where 19 detection positions d22 are arranged in the x-axis direction with the feature position P1 as the center is shown as an example. The detection of fluorescence or scattered light can be performed for a certain period of time at a single detection position d22. The fluorescence or scattered light detected within the certain period of time is integrated, converted into an electrical signal, and output to the control unit 103. The detection of fluorescence or scattered light can be performed by scanning the laser light in the x-axis direction and the z-axis direction and sequentially irradiating each detection position d22, and detecting the generated fluorescence or scattered light. Alternatively, fluorescence from each detection position d22 may be collectively detected by an area imaging device upon irradiation with the laser light.

[0148] (4-1-2) One-dimensional distribution parameter estimation step S302

[0149] In the one-dimensional distribution parameter estimation step S302, the control unit 10 assumes that the relationship between each detection position d22 and the integrated value or average value of the fluorescence detection intensity follows a one-dimensional distribution stored in a memory or the like. For example, when the detection intensity data shown in FIG. 12B is obtained, the control unit 103 can calculate the maximum value based on the least squares method, assuming the one-dimensional distribution as an N-th order polynomial model. By using an N-th order polynomial model rather than assuming a normal distribution for the one-dimensional distribution, it is possible to accurately cope with variations in the distribution of the optical profile due to design variations of each component constituting the apparatus for analyzing microparticles using the flow path. Also, the values used for the estimation in the one-dimensional distribution parameter estimation step S302 are not limited to the above integrated value and average value. As the values used for the estimation, for example, the median of the detection intensity or the number of events (particularly, the number of microparticles flowing during a certain period of time) may be used.

[0150] Here, when the distribution is an N-th order polynomial model, the higher the order, the higher the accuracy. However, if it is too high, it is likely to be affected by errors in the detection intensity at each detection position d22. Therefore, for example, it is preferable to set it to the fourth order.

[0151] (4-1-3) Maximum position movement step S303

[0152] In the maximum value position movement step S303, the control unit 103 outputs a movement signal to the control unit 103 to a position P2 where the integrated value or average value of the detection intensity is larger, preferably the maximum value, in the one-dimensional distribution assumed in the one-dimensional parameter estimation step S302. As a result, as shown in FIG. 12C, the control unit 103 moves the microchip 150 for microparticle separation so that the characteristic position P1 moves to the position P2. Note that the position P2 shown in FIG. 12C is shown as an example where it is on the detection position d22, but the position P2 may be between the two detection positions d22.

[0153] As described above, in the first fine adjustment step S300, the position of the flow path after the adjustment step can be adjusted with higher accuracy. In particular, the position adjustment of the microchip 150 is performed based on the detection intensity of the detection positions d22 arranged in one direction. Therefore, the number of data to be detected can be reduced as compared with performing position adjustment based on the intensities of detection positions arranged in a plurality of directions, for example, in a lattice shape. Accordingly, even if the number of arrangements is increased and the data accuracy is improved by narrowing the interval between the detection positions d22, an increase in the data detection time can be suppressed as compared with the case of acquiring data two-dimensionally.

[0154] (4-2) Second fine adjustment step FIG. 13 is a diagram for explaining the control in the second fine adjustment step S400. The second fine adjustment step S400 includes, for example, as shown in FIG. 17, a linear signal acquisition step S401, a one-dimensional parameter estimation step S402, and a maximum position movement step S403.

[0155] As shown in FIGS. 13A to C, the control in this step S23 is substantially the same as the control in the first fine adjustment step S300 described with reference to FIGS. 12A to C, except that, instead of moving the characteristic position P1 from P1 to P2 in the x-axis direction based on the detection intensity of the detection positions d22 arranged in the x-axis direction, the characteristic position P1 is moved from the position P2 to the position P3 in the z-axis direction based on the detection intensity of the detection positions d23 arranged in the z-axis direction with the position P2 as the center. Therefore, the description thereof is omitted here.

[0156] In the above description, in the first fine adjustment step S300, position adjustment is performed in the x-axis direction (the width direction of the flow path), and in the second fine adjustment step S400, position adjustment is performed in the z-axis direction (the optical axis direction). Regarding the additional position adjustment step, position adjustment may be performed in the z-axis direction in the first fine adjustment step S300, and position adjustment may be performed in the x-axis direction in the second fine adjustment step S400.

[0157] As described above, in the linear signal acquisition step S301 of the first fine adjustment step S300, fluorescence or scattered light is detected from a plurality of detection positions d22 arranged in the width direction (x-axis direction) of the flow path, with the adjusted characteristic position in the above-mentioned "(3-3) adjustment step" as the center. Here, the center may be shifted in the z-axis direction. For example, it may be shifted from the position in the z direction where the focus index is maximum to the position in the z direction where the signal from the microparticles (such as beads) is maximum. For example, the position in the z direction where the focus index is maximum in the moving step is a position specified based on image information regarding the structure of the flow path, such as the wall (e.g., the wall surface) or corner of the flow path. On the other hand, the position where the microparticles (such as cells or beads) flow in the microparticle sorting operation may be the center of the flow path. Therefore, an offset may occur between the position in the z direction where the focus index is maximum and the center of the flow path. For example, as shown in FIG. 19, the position in the z-axis direction where the maximum value is obtained for the plot S2 of the focus index in the z-axis direction obtained based on the autofocus function may be different from the position in the z-axis direction where the maximum value is obtained for the plot S1 of the optical signal generated by irradiating the microparticles with light. Therefore, in the linear signal acquisition step S301, the position adopted as the center may be the fluorescence or scattered light detected from a plurality of detection positions d22 arranged in the x-axis direction with the position shifted in the z-axis direction from the characteristic position by such a difference as the center. By performing such a shift, in the first fine adjustment step, the flow path can be moved to a more appropriate position.

[0158] (5) Another example of a microparticle analyzer that executes the position adjustment method of the present technology

[0159] The position adjustment method of the present technology may be executed by a microparticle analyzer (for example, a flow cytometer) that forms droplets and analyzes and / or separates microparticles. For example, the flow cell or the flow path in the microchip used in the apparatus may be the flow path whose position is adjusted in the position adjustment method of the present technology. Hereinafter, a configuration example of the apparatus will be described.

[0160] FIG. 14 shows a schematic diagram for explaining the configuration of a microparticle analyzer 1 (hereinafter also referred to as "flow cytometer 1") that forms droplets and analyzes and / or separates microparticles. FIGS. 15 and 16 show an example of a microchip 2 that can be mounted on the flow cytometer 1. FIG. 15A shows a top view, and B shows a cross-sectional schematic view corresponding to the P-P cross section in A. FIG. 16 is a diagram schematically explaining the configuration of the orifice 21 of the microchip 2, where A shows a top view, B shows a cross-sectional view, and C shows a front view. FIG. 16B corresponds to the P-P cross section in FIG. 15A.

[0161] (5-1) Irradiation detection unit The flow cytometer 1 includes an irradiation detection unit including a light source 61 that irradiates the microchip 2 with a laser L1 and a detector 62 that detects detection target light generated by the irradiation of the laser L1. The irradiation direction (optical axis of the laser L1) of the laser L1 with respect to the microchip 2 is shown in the positive Z-axis direction in FIG. 1. The light source 61 may be an LD, an LED, or the like.

[0162] The laser L1 is irradiated onto cells flowing through the sample flow path 22 of the microchip 2. The detector 62 detects scattered light of the laser L1 by the cells and fluorescence generated by excitation of the cells or fluorescent dyes labeled on the cells by the laser L1. In FIG. 14, fluorescence generated from cells flowing through the sample flow path 22 is indicated by coincidence F1.

[0163] The irradiation detection unit includes an irradiation system composed of a condenser lens, a dichroic mirror, a band-pass filter, etc. for guiding and condensing the laser L1 emitted from the light source 61 onto the cells. Further, the irradiation detection unit is constituted by a detection system that condenses the detection target light generated from the cells by the irradiation of the laser L1 and guides it to the detector 62. The detection system is constituted by, for example, a PMT (photo multiplier tube), an area imaging element such as a CCD or a CMOS element, etc.

[0164] The detection target light detected by the detection system of the irradiation detection unit is light generated from the cells by the irradiation of the laser L1, and can be, for example, forward scattered light, side scattered light, scattered light such as Rayleigh scattering and Mie scattering, fluorescence, etc. The fluorescence may be generated from the cells or fluorescent dyes labeled on the cells. These detection target lights are converted into electrical signals and used for determining the optical characteristics of the cells and the automatic adjustment of the optical position described later.

[0165] (5-2) Position adjustment unit The flow cytometer 1 includes a position adjustment unit 9 that changes the relative position of the microchip 2 with respect to the irradiation detection unit. The position adjustment unit 9 moves the position of the microchip 2 and / or the position of the irradiation detection unit on a vertical plane (XY plane) with respect to the optical axis of the laser L1. Thereby, the position adjustment unit 9 adjusts the position of the microchip 2 with respect to the optical axis of the laser L1 and optimizes it so that the laser L1 irradiates the cell flow position in the sample flow path 22.

[0166] The position adjustment unit 9 only needs to be able to move at least one of the position of the microchip 2 or the position of the irradiation detection unit including the light source 61 and the detector 62 in the X-axis direction and the Y-axis direction. The position adjustment unit 9 is constituted by, for example, a stepping motor, etc. Note that the position adjustment unit 9 may also move the relative position of the microchip 2 with respect to the irradiation detection unit in the Z-axis direction (the focal direction of the laser L1).

[0167] (5-3) Vibration element The flow cytometer 1 includes a vibration element 3 that applies vibration to the orifice 21 formed in the microchip 2 to atomize and discharge the laminar flow of the sample liquid containing cells and the sheath liquid discharged from the orifice 21. The vibration element 3 can be, for example, a piezo element. The discharged droplets become a fluid stream S and are ejected in the positive direction of the Y-axis of the arrow in the figure. In the flow cytometer 1, the microchip 2 is detachably mounted.

[0168] In the flow cytometer 1, the vibration element 3 may be configured integrally with the microchip 2, or may be disposed on the device side so as to be in contact with the mounted microchip 2.

[0169] (5-4) Charging section The droplets discharged from the orifice 21 are given positive or negative charges by the charging section 41. The charging of the droplets is performed by an electrode 42 that is electrically connected to the charging section 41 and inserted into the sample inlet 23 provided in the microchip 2. The electrode 42 may be inserted at any location of the microchip 2 so as to be in electrical contact with the sample liquid or the sheath liquid fed through the flow path.

[0170] In the flow cytometer 1, by synchronizing the frequency of the drive voltage of the vibration element 3 and the switching timing of the voltage (charge voltage) of the charging section 41, either positive or negative charge is given to a part of the droplets discharged from the orifice 21. Some droplets may not be given a charge and may be uncharged.

[0171] (5-5) Deflection plates Furthermore, the flow cytometer 1 includes a pair of deflection plates 51, 52 arranged to face each other with the fluid stream S interposed therebetween. The deflection plates 51, 52 change the traveling direction of each droplet in the fluid stream S by the electrical force acting between them and the charge given to the droplets. The deflection plates 51, 52 may be electrodes commonly used. In FIG. 14, the facing direction of the polarizing plates 51, 52 is indicated by the X-axis direction.

[0172] (5-6) Recovery container The fluid stream that has passed between the deflection plates 51 and 52 is received by either the recovery container 81, the recovery container 82, or the recovery container 83. For example, when the deflection plate 51 is positively charged and the deflection plate 52 is negatively charged, the droplets negatively charged by the charging unit 41 are recovered in the recovery container 82, and the positively charged droplets are recovered in the recovery container 83, respectively. Also, the droplets not charged by the charging unit 41 fly straight without being affected by the electrical force from the deflection plates 51 and 52 and are recovered in the recovery container 81. In the flow cytometer 1, by controlling the traveling direction of each droplet according to the characteristics of the microparticles (e.g., biological particles, particularly cells) contained in the droplet, the target microparticles having desired characteristics and the non-target microparticles other than them can be recovered in separate recovery containers.

[0173] The recovery containers 81, 82, and 83 may be plastic tubes or glass tubes commonly used for experiments. These recovery containers are preferably arranged in the flow cytometer 1 in an exchangeable manner. Also, a drainage path for the recovered droplets may be connected to the recovery container that receives the non-target microparticles among the recovery containers. Note that in the flow cytometer 1, the number of recovery containers to be arranged is not particularly limited. When arranging more than three recovery containers, each droplet is induced and recovered in one of the recovery containers depending on the presence or absence and magnitude of the electrical force between the deflection plates 51 and 52.

[0174] (5-7) Control unit, etc. In addition to the above-described configuration, the flow cytometer 1 includes a data analysis unit for determining the optical characteristics of cells, a tank unit for storing the sample liquid and the sheath liquid, and a control unit 10 for controlling each of these components, which are provided in a normal flow cytometer. The control unit 10 includes the movement control unit 108 and the positional relationship adjustment unit 109 described in (3) above.

[0175] The control unit 10 can be configured by a general-purpose computer including a CPU, a memory, a hard disk, etc. In addition to the OS, the hard disk of the control unit 10 may store a program for causing the flow cytometer 1 to execute the position adjustment method of the present technology and a program for causing the flow cytometer 1 to execute a microparticle analysis operation performed after the position adjustment method of the present technology. The program may not be stored in the hard disk provided in the flow cytometer 1. In this case, it may be recorded on a recording medium such as a microSD memory card, an SD memory card, or a flash memory. The control unit 10 can cause the flow cytometer 1 to execute the position adjustment method of the present technology according to the program.

[0176] The control unit 10 can output a movement signal to the position adjustment unit 9 to a position where the integrated value or average value of the detected intensity of the light generated from the microchip by the irradiation of the laser L1 is larger (preferably the maximum value) and the variation is smaller among the preset regions.

[0177] (5-8) Microchip The microchip 2 is formed by bonding substrate layers 2a and 2b in which a sample flow path 22 is formed. The formation of the sample flow path 22 in the substrate layers 2a and 2b can be performed by injection molding of a thermoplastic resin using a mold. As the thermoplastic resin, plastics known as materials for conventional microchips such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS) can be adopted.

[0178] The sample liquid is introduced into the sample inlet 23, merges with the sheath liquid introduced into the sheath inlet 24, and is fed through the sample flow path 22. The sheath liquid introduced from the sheath inlet 24 is divided and fed in two directions, and then merges with the sample liquid at the confluence with the sample liquid introduced from the sample inlet 23, merging with the sample liquid so as to sandwich the sample liquid from two directions. As a result, at the confluence, a three-dimensional laminar flow is formed in which the sample liquid laminar flow is positioned at the center of the sheath liquid laminar flow.

[0179] Reference numeral 25 indicates a suction flow path for applying a negative pressure into the sample flow path 22 to temporarily reverse the flow and eliminate clogging or bubbles when clogging or bubbles occur in the sample flow path 22. At one end of the suction flow path 25, a suction outlet 251 connected to a negative pressure source such as a vacuum pump is formed, and the other end is connected to the sample flow path 22 at the communication port 252.

[0180] The three-dimensional laminar flow is narrowed in the constriction portions 261 (see FIG. 15), 262 (see FIG. 16) formed so that the area of the vertical cross section with respect to the liquid feeding direction gradually or stepwise decreases from the upstream to the downstream in the liquid feeding direction. Thereafter, the three-dimensional laminar flow is discharged from the orifice 21 provided at one end of the flow path.

[0181] Between the constriction portion 261 and the constriction portion 262 of the sample flow path 22, the characteristics of the cells are detected. By the irradiation detection unit, the laser L1 is irradiated onto the cells that are arranged in a line at the center of the three-dimensional laminar flow and fed through the sample flow path 22, and the fluorescence F1 and scattered light generated from the cells are detected (see FIG. 15). The position adjustment method of the present technology may be performed on the flow path between the constriction portion 261 and the constriction portion 262 in the sample flow path 22.

[0182] The connection portion of the sample flow path 22 to the orifice 21 is a straight portion 27 formed linearly. The straight portion 27 functions to eject the fluid stream S straight in the positive Y-axis direction from the orifice 21.

[0183] The three-dimensional laminar flow discharged from the orifice 21 is atomized by the vibration applied to the orifice 21 by the vibration element 31 and ejected as a fluid stream S (see FIG. 14). The orifice 21 opens in the end face direction of the substrate layers 2a and 2b, and a notch 211 is provided between the opening position thereof and the substrate layer end face. The notch 211 is formed by cutting out the substrate layers 2a and 2b between the opening position of the orifice 21 and the substrate end face so that the diameter L of the notch 221 is larger than the opening diameter l of the orifice 21 (see FIG. 16C). The diameter L of the notch 211 is preferably formed to be more than twice as large as the opening diameter l of the orifice 21 so as not to impede the movement of the droplets discharged from the orifice 21.

[0184] (5-9) Imaging optical system The microparticle analyzer 1 includes an imaging optical system (not shown) that images the flow path between the constriction portion 261 and the constriction portion 262 in the sample flow path 22. As described in the above “(2-4) Configuration example of the optical system”, the imaging optical system may include an illumination device 370 and an imaging element 371. The description in the above “(2-4) Configuration example of the optical system” also applies to the imaging optical system included in the microparticle analyzer 1.

[0185] 2. Second Embodiment (Microparticle Analyzer)

[0186] The present technology also provides a microparticle analyzer including an imaging optical system that images a flow path through which microparticles can flow while moving the flow path in the optical axis direction at a plurality of positions in the optical axis direction, a movement control unit that moves the flow path in the optical axis direction based on a focus index for each of a plurality of images captured by the imaging optical system, and a positional relationship adjustment unit that adjusts the positional relationship in a direction perpendicular to the optical axis direction between a feature position specified from an image of the flow path at the moved position and a reference position.

[0187] The microparticle analyzer of the present technology can execute the position adjustment method described in the above "1. First Embodiment (Position Adjustment Method)" by including the imaging optical system, the movement control unit, and the position relationship adjustment unit. Therefore, in the microparticle analyzer of the present technology, the number of microparticles used for adjusting the position of the flow path where light irradiation for analyzing microparticles is performed can be reduced.

[0188] The microparticle analyzer of the present technology may have a configuration as described in the above "1. First Embodiment (Position Adjustment Method)", and the description thereof also applies to the present embodiment.

[0189] 3. Third Embodiment (Program)

[0190] The present technology also provides a program for causing a microparticle analyzer to execute the position adjustment method of the present technology. The position adjustment method is as described in the above "1. First Embodiment (Position Adjustment Method)". The program can be realized, for example, by the microparticle analyzer 100 described in the above "1. First Embodiment (Position Adjustment Method)", particularly by the control unit 103. Further, the program can be realized, for example, by the microparticle analyzer 1 described in the above "1. First Embodiment (Position Adjustment Method)", particularly by the control unit 10.

[0191] Note that the present technology can also have the following configuration. 〔1〕An imaging step of imaging the flow path at a plurality of positions in the optical axis direction while moving the flow path through which microparticles can flow in the optical axis direction, A moving step of moving the flow path in the optical axis direction based on a focus index for each of the plurality of images obtained in the imaging step, and An adjustment step of specifying a characteristic position of the flow path from an image of the flow path at the position after movement in the moving step and adjusting the positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction. A position adjustment method including the above steps. The position adjustment method according to [1], wherein [2] the focus index is an index indicating whether the flow path is in focus. The position adjustment method according to [1] or [2], wherein [3] the focus index is a focus index obtained using an autofocus function. The position adjustment method according to [3], wherein [5] the autofocus function is a function based on image difference. The position adjustment method according to [3] or [4], wherein [7] the autofocus function is a Brenner function. The position adjustment method according to any one of [1] to [5], wherein [6] the flow path is used to irradiate laser light onto microparticles flowing through the flow path and analyze the microparticles based on the light generated by the irradiation. The position adjustment method according to [6], wherein

[11] the direction of the optical axis of the laser light is substantially the same as the optical axis direction in the imaging step. 〔8〕The movement step includes an image identification step of identifying an image that gives a focus index satisfying a predetermined criterion from the plurality of acquired focus indices, and in the movement step, the flow path is moved to the position where the image identified in the image identification step was captured. The position adjustment method according to any one of [1] to [7]. 〔9〕The movement step includes an image identification step of identifying an image that gives the maximum or minimum focus index from the plurality of acquired focus indices, and in the movement step, the flow path is moved to the position where the image identified in the image identification step was captured. The position adjustment method according to any one of [1] to [8]. The position adjustment method according to any one of [1] to [9], wherein

[10] the characteristic position is identified based on the position of the wall defining the flow path. The position adjustment method according to any one of [1] to

[10] , wherein

[11] the characteristic position is substantially the central position in the width direction of the flow path. 〔12〕The position adjustment method according to any one of 〔1〕 to 〔11〕, wherein the reference position is a position passing through the optical axis of the laser light irradiated on the microparticles flowing through the flow path. 〔13〕The position adjustment method according to any one of 〔1〕 to 〔12〕, wherein the positional relationship is a positional relationship in the width direction of the flow path. 〔14〕The position adjustment method according to any one of 〔1〕 to 〔13〕, wherein in the adjustment step, the flow path is moved in the width direction of the flow path. 〔15〕The flow path is used for irradiating laser light on the microparticles flowing through the flow path and analyzing the microparticles based on the light generated by the irradiation. The position adjustment method according to any one of 〔1〕 to 〔14〕, wherein in the adjustment step, the irradiation position of the laser light is adjusted. 〔16〕The method further includes an additional position adjustment step of irradiating laser light on the microparticles flowing through the flow path and further adjusting the position of the flow path based on the light generated by the irradiation. The position adjustment method according to any one of 〔1〕 to 〔15〕. 〔17〕The position adjustment method according to any one of 〔1〕 to 〔16〕, wherein the microparticles are biological particles. 〔18〕An imaging optical system that images the flow path at a plurality of positions in the optical axis direction while moving the flow path through which microparticles can flow in the optical axis direction. A movement control unit that moves the flow path in the optical axis direction based on a focus index for each of a plurality of images captured by the imaging optical system, and A position relationship adjustment unit that adjusts the position relationship in a direction perpendicular to the optical axis direction between a feature position specified from an image of the flow path at the moved position and a reference position. A microparticle analyzer including the above. 〔19〕The microparticle analyzer An irradiation optical system that irradiates laser light on the microparticles flowing through the flow path, and A light detection system that detects the light generated by the irradiation of the laser light. further includes The direction of the optical axis of the laser light is substantially the same as the direction of the optical axis of the imaging optical system. The microparticle analyzer according to

[18] . 〔20〕The microparticle analyzer An irradiation optical system that irradiates laser light onto the microparticles flowing through the flow path, and A light detection system that detects the light generated by the irradiation of the laser light further includes One or both of the irradiation optical system and the light detection system share at least one of the optical components included in the imaging optical system. The microparticle analyzer according to

[18] or

[19] . 〔21〕An imaging step of imaging the flow path at a plurality of positions in the optical axis direction while moving the flow path through which microparticles can flow in the optical axis direction, A moving step of moving the flow path in the optical axis direction based on a focus index for each of the plurality of images obtained in the imaging step, and An adjustment step of specifying a characteristic position of the flow path from an image of the flow path at the position after movement in the moving step, and adjusting a positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction A program for causing a microparticle analyzer to execute.

Explanation of Signs

[0192] 100 Microparticle analyzer 103 Control unit 108 Movement control unit 109 Positional relationship adjustment unit 150 Microchip for microparticle analysis

Claims

1. an imaging step of imaging the flow path, through which microparticles can flow, at a plurality of positions in the optical axis direction while moving the flow path in the optical axis direction; a moving step of moving the flow path in the optical axis direction based on a focus index for each of a plurality of images obtained in the imaging step; and an adjustment step of identifying a characteristic position of the flow path from an image of the flow path at a position after the movement in the movement step, and adjusting a positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction; Including, The characteristic position is identified based on an average pixel value acquired for each position in a width direction of an image of the flow path at a position after the movement in the moving step. How to adjust the position.

2. The position adjustment method according to claim 1 , wherein the focus index is an index that indicates whether the flow channel is in focus.

3. The position adjustment method according to claim 1 , wherein the focus index is obtained by using an autofocus function.

4. The alignment method of claim 3 , wherein the autofocus function is an image differencing based function.

5. The alignment method of claim 3 , wherein the autofocus function is a Brenner function.

6. 2. The position adjustment method according to claim 1, wherein the flow channel is used for irradiating a microparticle flowing through the flow channel with a laser light and analyzing the microparticle based on the light generated by the irradiation.

7. The position adjustment method according to claim 6 , wherein a direction of an optical axis of the laser light is substantially the same as a direction of an optical axis in the imaging step.

8. the moving step includes an image specifying step of specifying an image that gives a focus index that satisfies a predetermined criterion from the plurality of focus indices acquired, In the moving step, the flow path is moved to a position where the image specified in the image specifying step was captured. The position adjustment method according to claim 1 .

9. the moving step includes an image specifying step of specifying an image giving a maximum or minimum focus index from the plurality of focus indices acquired, In the moving step, the flow path is moved to a position where the image specified in the image specifying step was captured. The position adjustment method according to claim 1 .

10. The position adjusting method according to claim 1 , wherein the characteristic position is a substantially central position in a width direction of the flow channel.

11. The position adjustment method according to claim 1 , wherein the reference position is a position where an optical axis of a laser beam irradiated onto the microparticle flowing through the flow channel passes through.

12. The position adjusting method according to claim 1 , wherein the positional relationship is a positional relationship in a width direction of the flow channel.

13. The position adjusting method according to claim 1 , wherein in the adjusting step, the flow path is moved in a width direction of the flow path.

14. the flow channel is used for irradiating a microparticle flowing through the flow channel with a laser light and analyzing the microparticle based on the light generated by the irradiation, The position adjusting method according to claim 1 , wherein in the adjusting step, an irradiation position of the laser light is adjusted.

15. Further comprising an additional position adjustment step of irradiating a laser beam onto the microparticles flowing through the flow path and further adjusting the position of the flow path based on the light generated by the irradiation. The position adjustment method according to claim 1 .

16. The position adjustment method according to claim 1 , wherein the microparticles are biological particles.

17. an imaging optical system that images a flow path through which microparticles can flow at a plurality of positions in an optical axis direction while moving the flow path in the optical axis direction; a movement control unit that moves the flow path in the optical axis direction based on a focus index for each of a plurality of images captured by the imaging optical system; and a positional relationship adjustment unit that adjusts a positional relationship between a characteristic position identified from an image of the flow channel at a position after the movement and a reference position in a direction perpendicular to the optical axis direction; Including, The characteristic position is identified based on an average pixel value acquired for each position in a width direction of an image of the flow channel at a position after the movement by the movement control unit. Microparticle analyzer.

18. The microparticle analysis device comprises: an irradiation optical system that irradiates the microparticles flowing through the flow path with laser light; and a light detection system for detecting light generated by the irradiation of the laser light; Further comprising: The direction of the optical axis of the laser light is substantially the same as the optical axis direction of the imaging optical system. The microparticle analysis device according to claim 17.

19. The microparticle analysis device comprises: an irradiation optical system that irradiates the microparticles flowing through the flow path with laser light; and a light detection system for detecting light generated by the irradiation of the laser light; Further comprising: One or both of the irradiation optical system and the light detection system share at least one optical component included in the imaging optical system. The microparticle analysis device according to claim 17.

20. an imaging step of imaging the flow path, through which microparticles can flow, at a plurality of positions in the optical axis direction while moving the flow path in the optical axis direction; a moving step of moving the flow path in the optical axis direction based on a focus index for each of a plurality of images obtained in the imaging step; and an adjustment step of identifying a characteristic position of the flow path from an image of the flow path at a position after the movement in the movement step, and adjusting a positional relationship between the characteristic position and a reference position in a direction perpendicular to the optical axis direction; The microparticle analysis device executes the above. The characteristic position is identified based on an average pixel value acquired for each position in a width direction of an image of the flow path at a position after the movement in the moving step. program.

Images