Microparticle analysis device and microparticle analysis method

Abstract

To provide a technique for stabilizing brake-off timings with respect to a microparticle analysis device and a microparticle analysis method.SOLUTION: A microparticle analysis device or the like comprises at least: a flow path in which a fluid including a sample flow containing microparticles and a sheath flow flowing to contain the sample flow flows; a droplet formation section that forms a droplet in the fluid by imparting vibration to the fluid using a vibration element 13; an electric charge application section 11 that applies electric charge to a droplet containing the microparticles; an imaging section that obtains a photograph of a phase of a certain time; and a control section 4 that controls timing at which the droplet brakes off on the basis of the photograph.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present technology relates to a microparticle analysis device and a microparticle analysis method. [Background technology]

[0002] Currently, a technology called flow cytometry is used to analyze microparticles related to living organisms such as cells and microorganisms. Flow cytometry is an analytical technique that analyzes and separates microparticles by irradiating light onto microparticles that flow as if contained in a sheath flow that is sent through a flow channel formed in a flow cell or microchip, and detecting the fluorescence and scattered light emitted from each microparticle. The device used in flow cytometry is called a flow cytometer (sometimes called a "cell sorter").

[0003] In this flow cytometer, a vibration element is generally provided in a part of the flow path through which the microparticles wrapped in a sheath flow flow. This vibration element vibrates a part of the flow path, and the fluid discharged from the discharge port of the flow path is continuously turned into droplets. The flow cytometer employs a configuration in which a predetermined electric charge is applied to the droplets containing the microparticles, and based on this electric charge, the direction of travel of the droplets is changed by a deflection plate or the like, and only the target microparticles are collected in a predetermined container or a predetermined location on a plate.

[0004] In a flow cytometer, a technique for controlling the stable formation of droplets is one of the important factors for improving the accuracy of analysis. Here, it is known that if the formation of droplets is unstable, such as the timing of break-off, when the fluid discharged from the outlet of the flow channel turns into droplets, is unstable, the time at which the droplets are charged also becomes unstable, and as a result, the sorting of microparticles also becomes unstable. However, it is difficult to control the formation of droplets because multiple factors such as flow rate, environmental conditions such as temperature and humidity, and the size of the microparticles are involved.

[0005] In response to this, for example, Patent Document 1 discloses a technique for stabilizing the timing of break-off. In this technique, the magnitude of vibration is controlled according to the distance from the break-off point to the first satellite. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2015-152439 A Summary of the Invention [Problem to be solved by the invention]

[0007] However, the reality is that further development of technology for stably forming droplets is desired.

[0008] Therefore, a main object of the present technology is to provide a technology for stabilizing the timing of break-off. [Means for solving the problem]

[0009] In this technology, first, a flow path for a fluid including a sample flow containing microparticles and a sheath flow flowing so as to contain the sample flow, a droplet forming section for forming droplets in the fluid by applying vibration to the fluid using a vibration element, a charge charging section for charging the droplets containing the microparticles, an imaging section for taking a photograph of the phase at a certain time, and a method for detecting the phase based on the photograph. and a control unit that controls the timing at which the droplets break off. In the microparticle analysis device according to the present technology, the control unit may adjust the voltage of the vibration element based on the timing at which the droplets break off, which is specified by the multiple photographs. In addition, in the microparticle analysis device according to the present technology, the control unit may acquire sequential photographs of phases within one period, and identify the phase of timing when the droplet breaks off in the sequential photographs. In this case, the control unit may acquire sequential photographs further divided into phases before and after the phase of timing when the droplet breaks off, and identify the phase of timing when the droplet breaks off in the divided sequential photographs. In this case, the imaging unit may include a strobe, and the identification may be performed using sequential phase photographs obtained by changing the strobe emission start time of the strobe. Furthermore, in the microparticle analysis device related to the present technology, the control unit may control the phase of the timing at which the droplet breaks off to be a phase corresponding to the opposite direction of the phase at which the droplet is charged, and the control unit may also control the phase at which the microparticle is charged to be between the phase at which the droplet preceding the droplet breaks off from the liquid column and the phase at which a satellite of the droplet breaks off from the liquid column.

[0010] The present technology also provides a microparticle analysis method that includes at least a droplet formation step of forming droplets in a fluid consisting of a sample flow containing microparticles and a sheath flow flowing to encapsulate the sample flow by applying vibrations using a vibration element to form droplets in the fluid, a charge charging step of charging the droplets encapsulating the microparticles, an imaging step of obtaining a photograph of a phase at a certain time, and a control step of controlling the timing at which the droplets break off based on the photograph. In the microparticle analysis method according to the present technology, in the control step, a voltage of the vibration element may be adjusted based on a timing at which the droplet breaks off, the timing being specified by the plurality of photographs.

[0011] In the present technology, the term "microparticles" may broadly include biologically relevant microparticles such as cells, microorganisms, and ribosomes, as well as synthetic particles such as latex particles, gel particles, and industrial particles.

[0012] The bio-related microparticles include chromosomes, ribosomes, mitochondria, organelles (cell organelles), and the like that constitute various cells. The cells include animal cells (e.g., blood cells, etc.) and plant cells. The microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, the bio-related microparticles also include bio-related polymers such as nucleic acids, proteins, and complexes thereof. The industrial particles may be, for example, organic or inorganic polymer materials, metals, and the like. The organic polymer materials include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, and the like. The inorganic polymer materials include glass, silica, magnetic materials, and the like. The metals include gold colloids, aluminum, and the like. The shape of these microparticles is generally spherical, but in the present technology, they may be non-spherical, and their size, mass, and the like are not particularly limited. Effect of the Invention

[0013] According to the present technology, it is possible to provide a technology for stabilizing the timing of break-off. Note that the effects described herein are not necessarily limited to those described herein and may be any of the effects described in this disclosure. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing an example of an embodiment of a microparticle analysis device 1 according to the present technology. [Diagram 2] 2A and 2B are schematic diagrams showing an example of the configuration of a microchip 2 that can be used in the microparticle analysis device 1 of FIG. [Diagram 3] 3A to 3C are schematic diagrams showing an example of the configuration of the orifice 21 of the microchip 2 that can be used in the microparticle analysis device 1 of FIG. [Figure 4] FIG. 4A is a photograph substituting a drawing showing the state of the stream when no splash is being generated, and FIG. 4B and FIG. 4C are photographs substituting a drawing showing the state of the stream when splash is being generated. [Diagram 5]5A to 5D are diagrams illustrating a method of control example 1. [Figure 6] 7 is a drawing substitute photograph showing a series of photographs in which one cycle of FIG. 6 is divided into 16 equal parts. [Figure 7] 7A to 7C are diagrams illustrating a method of control example 2. [Figure 8] 8A to 8C are diagrams illustrating a method of control example 3. [Figure 9] 9A to 9D are diagrams illustrating a method of control example 4. [Figure 10] FIG. 10 is a flowchart showing an example of a flow of microparticle analysis using the microparticle analysis method according to the present technology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, preferred embodiments of the present technology will be described with reference to the drawings. The embodiment described below is an example of a typical embodiment of the present technology, and is not intended to narrow the scope of the present technology. The description will be given in the following order. 1. Microparticle analyzer 1 (1) Flow path (1-1) Microchip 2 (2) Droplet forming part (3) Electric charge unit 11 (4) Imaging unit 3 (5) Control Unit 4 [Control example 1] [Control example 2] [Control example 3] [Control example 4] (6) Detection unit 5 (7) Deflection plates 61, 62 (8) Collection containers 71-73 (9) Analysis section 8 (10) Storage section 9 (11) Display section 10 (12) Input section (13)Other 2. Microparticle analysis method (1) Droplet formation step (2) Charge step (3) Imaging step (4) Control step [An example of the flow of microparticle analysis]

[0016] 1. Microparticle analyzer 1 1 is a schematic diagram showing an example of an embodiment of a microparticle analysis device 1 according to the present technology. The microparticle analysis device 1 (flow cytometer) according to the present technology at least includes a flow path, a droplet formation unit, a charge charge unit 11, an imaging unit 3, and a control unit 4. In addition, as necessary, a detection unit 5, deflection plates 61, 62, collection containers 71 to 73, an analysis unit 8, a storage unit 9, a display unit 10, The device may also include an input unit, etc. Each unit will be described in detail below.

[0017] (1) Flow path A fluid consisting of a sample flow containing microparticles and a sheath flow that flows so as to enclose the sample flow passes through the flow path. This flow path may be provided in advance in the microparticle analysis device 1 according to the present technology, but it is also possible to install a disposable chip or the like provided with the flow path in the microparticle analysis device 1 and perform analysis or separation.

[0018] The shape of the flow channel that can be used in the microparticle analysis device 1 according to the present technology is not particularly limited and can be freely designed. In the present technology, it is particularly preferable to use a flow channel formed in a two-dimensional or three-dimensional substrate such as plastic or glass.

[0019] In addition, the width, depth, and cross-sectional shape of the flow channel are not particularly limited as long as they can form a laminar flow, and can be freely designed. For example, a micro flow channel with a width of 1 mm or less can be used in the microanalysis measurement device 1 according to the present technology. In particular, a micro flow channel with a width of about 10 μm or more and 1 mm or less is preferably used in the microparticle analysis device 10 according to the present technology.

[0020] (1-1) Microchip 2 Fig. 2 is a schematic diagram showing an example of the configuration of a microchip 2 usable in the microparticle analysis device 1 of Fig. 1, and Fig. 3 is a schematic diagram showing an example of the configuration of an orifice 21 of a microchip 2 usable in the microparticle analysis device 1 of Fig. 1. A of Fig. 2 is a schematic top view, and B of Fig. 2 is a schematic cross-sectional view corresponding to the PP cross section in A. Also, A of Fig. 3 is a top view, B of Fig. 3 is a cross-sectional view, and C of Fig. 3 is a front view. Note that B of Fig. 3 corresponds to a part of the PP cross section in A of Fig. 2.

[0021] The microchip 2 is formed by bonding together substrate layers 2a and 2b in which a sample flow path 22 is formed. The sample flow path 22 can be formed in the substrate layers 2a and 2b by injection molding of a thermoplastic resin using a mold. The thermoplastic resin may be a plastic known as a material for conventional microchips, such as polycarbonate, polymethylmethacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS).

[0022] The microchip 2 is also formed with a sample inlet 23 for introducing a sample containing microparticles from a liquid sending connector, a sheath inlet 24 for introducing a sheath liquid, and a sample flow path 22 for introducing a sample flow and merging with the sheath liquid. The sheath liquid introduced from the sheath inlet 24 is sent in two directions, and then merges with the sample liquid introduced from the sample inlet 23 at the junction, sandwiching the sample liquid from two directions. As a result, a three-dimensional laminar flow is formed at the junction, with the sample liquid laminar flow located in the center of the sheath liquid laminar flow.

[0023] 2A indicates a suction channel for applying negative pressure to the sample channel 22 to temporarily reverse the flow and eliminate clogging or air bubbles when the sample channel 22 is clogged or has air bubbles. One end of the suction channel 25 is formed with a suction outlet 251 connected to a negative pressure source such as a vacuum pump. The other end of the suction channel 25 is connected to the sample channel 22 at a communication port 252.

[0024] The three-dimensional laminar flow is narrowed in width at narrowing sections 261 (see FIG. 2) and 262 (see A and B in FIG. 3), which are formed so that the area of ​​the cross section perpendicular to the liquid sending direction gradually or stepwise decreases from the upstream to the downstream in the liquid sending direction. The fluid is discharged as a fluid stream from the orifice 21. In Fig. 1, the discharge direction of the fluid stream from the orifice 21 is indicated by the positive direction of the Y axis.

[0025] The connection portion of the sample flow channel 22 to the orifice 21 is a straight portion 27 formed in a linear shape. The straight portion 27 functions to eject the fluid stream from the orifice 21 straight in the positive direction of the Y axis.

[0026] The fluid stream ejected from the orifice 21 is turned into droplets by vibration applied to the orifice 21 by the vibration element 13 (described later) in accordance with a droplet frequency (DropletCLK). The orifice 21 opens toward the end faces of the substrate layers 2a and 2b, and a notch 211 is provided between the opening position and the substrate layer end faces. 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 faces such that the diameter L of the notch 21 is larger than the opening diameter l of the orifice 21 (see FIG. 3C). It is preferable that the diameter L of the notch 211 is formed to be at least twice as large as the opening diameter l of the orifice 21 so as not to impede the movement of the droplets ejected from the orifice 21.

[0027] (2) Droplet forming part The droplet forming section uses vibration element 13 to apply vibration to the fluid to form droplets in the fluid. Vibration element 13 is preferably provided so as to be in contact with the flow path, and more preferably provided near the fluid discharge port of the flow path as shown in Fig. 1. In particular, when microchip 2 is used, it is preferably provided near orifice 21 of microchip 2 described above.

[0028] The vibration element 13 is not particularly limited, and any known element can be freely selected and used. Specifically, it is, for example, a piezoelectric element. By adjusting the amount of liquid sent to the flow channel, the diameter of the discharge port, the vibration frequency of the vibration element 13, etc., and adjusting the size of the droplets, droplets containing a fixed amount of microparticles can be generated.

[0029] (3) Electric charge unit 11 The electric charge charging unit 11 charges the droplets containing the microparticles with a positive or negative charge. When the microchip 2 is used, an electric charge is applied to the droplets discharged from an orifice 21 formed in the microchip 2. The electric charge charging unit 11 is disposed upstream of the imaging unit 3 described later, for example, as shown in FIG. 1. The droplets are charged by an electrode 12 that is electrically connected to the electric charge charging unit 11 and inserted into a sample inlet 23 provided in the microchip 2. The electrode 12 may be inserted at any point in the microchip 2 so as to be in electrical contact with the sample liquid or sheath liquid being pumped through the flow path.

[0030] In the microparticle analysis device 1 according to the present technology, after a microparticle contained in a sample liquid is detected by a detection unit 5 described later, the electric charge charging unit 11 can charge a droplet containing the microparticle after a drop delay time has elapsed.

[0031] (4) Imaging unit 3 The imaging unit 3 takes a photograph of a phase (droplet image) at a certain time. Reference numeral 31 in FIG. 1 denotes a droplet camera 32, such as a CCD camera or a CMOS sensor, for capturing an image of a droplet discharged from the orifice 21 of the microchip 2. The droplet camera 32 is disposed downstream of the detection unit 5, which will be described later, and captures an image of at least a portion of the fluid. The droplet camera 32 is also capable of adjusting the focus of the captured image of the droplet. As a light source for capturing images in the droplet camera 32, for example, a strobe 31, which will be described later, is used. The imaging unit 3 can also obtain a plurality of the above-mentioned photographs, and the image capturing unit 3 can capture an image of the droplet at a constant interval. It is also possible to continuously acquire photographs within a certain period. The "constant period" here is not particularly limited, and may be one period, which will be described later, or multiple periods. In the case of multiple periods, each period may be continuous in time or may be discontinuous.

[0032] The image captured by the droplet camera 32 is displayed on a display unit such as a display, and can be used by the user to check the state of droplet formation in the orifice 21 (for example, the size, shape, intervals, etc. of the droplets).

[0033] The strobe 31 may be controlled by the control unit 4 described later. The strobe 31 is composed of an LED for capturing an image of a droplet and a laser L2 (e.g., a red laser light source) for capturing an image of a microparticle, and the light source to be used is switched by the control unit 4 depending on the purpose of capturing the image. The specific structure of the strobe 31 is not particularly limited, and one or more types of known circuits or elements can be selected and freely combined.

[0034] If you use an LED for strobe 31, the LED will light for one Droplet CLK cycle. This light emission occurs every Droplet CLK. This makes it possible to extract and acquire an image of a certain moment when droplets are formed. The droplet camera 32 captures images about 30 times per second, while the DropletCLK is about 10 kHz to 50 kHz, for example, but the present technology is not limited to this.

[0035] When the laser L2 is used for the strobe 31, the laser L2 emits light at a half cycle of DropletCLK or at a cycle shorter than that. In this case, the laser L2 is emitted after the light source lighting delay time set by the control unit 4 has elapsed only when the microparticle is detected by the detection unit 5, so that the fluorescence of the microparticle contained in the droplet can be obtained from the image. The droplet camera 32 captures images about 60 times per second, and by performing measurements so that the detection of microparticles and the laser L2 light source emission are several thousand times per second, an image of stable microparticles in which the fluorescence of several tens of microparticles has accumulated can be obtained. The emission time of the laser L2 may be a time that allows a stable microparticle image to be obtained.

[0036] (5) Control Unit 4 The control unit 4 controls the timing at which the droplets break off based on the photograph obtained by the imaging unit 3.

[0037] Conventionally, small droplets formed when a thin rod-shaped liquid column that is stretched to the rear after droplet ejection separates from the main droplet and nozzle due to surface tension are called "satellites." Since satellites cause fluctuations in droplet charge, control of the satellites has been an essential parameter for microparticle analysis devices, which require high accuracy in the deflection position of droplets.

[0038] The types of satellites are Slow Satellite (Back satellite), Infinity, and Fast Satellite (Forward Satellite). There are four types: Slow Satellite, Nonsatellite, and Slow Satellite. is when the bottom end of the satellite is cut off and then the top end of the satellite is cut off, Infinity is when the bottom and top ends of the satellite are cut off at the same time, Fastsatellite is when the top end of the satellite is cut off and then the bottom end of the satellite is cut off, and Nonsatellite is when the top end of the satellite is cut off and is absorbed before the bottom end is cut off.

[0039] If the timing of the satellite tearing off and the timing of the charge are not synchronized, this can be one of the causes of splashing. The target droplets have a certain amount of positive or negative charge, so they split off to the left and right, while the non-target droplets have no charge and so pass straight through the center.

[0040] However, if a discrepancy occurs in the charging timing, some of the target droplets are properly charged and some are not (satellites are not charged), so multiple left and right streams are detected, as shown in Figure 4 B. Also, some untargeted droplets are charged (satellites are charged), so multiple central streams are also detected, as shown in Figure 4 C.

[0041] Therefore, if sorting is performed while droplets are being generated, the cells will not be dropped in the desired location, resulting in a decrease in yield and recovery rate.In addition, unintended microparticles may be sorted, resulting in a decrease in purity.

[0042] Therefore, by using this technology, the control unit 4 controls the timing at which the droplets break off, thereby stabilizing the timing of break-off by preventing a discrepancy between the timing at which the satellites break off and the timing at which the electric charge is applied.

[0043] In addition, in the past, indices such as Gap length (the distance from the break-off point to the satellite), satellite length, and droplet length were used to stabilize the timing of break-off. In other words, they tried to control it by observing a part of the fluid that was not a constricted part just before it was turned into droplets, and indirectly capturing the change in the constricted part. However, such indirect indices are easily affected by the size deviation of microparticles in the sample liquid, changes in environmental temperature, etc., and if the relationship between the index and the constriction is broken by disturbance, there is a possibility that the performance cannot be maintained. Specifically, when large-sized microparticles enter, a force that disturbs the surface works according to the vibration intensity, making it easier for the break-off to shift, and the time until break-off mainly depends on the growth rate (a parameter related to the ease of constriction of droplets), which is linear with the change in the fluid surface tension and the change in fluid density, and if physical quantities such as temperature vary, it affects the state of the fluid and changes the time until break-off.

[0044] However, with this technology, the break-off point can be directly monitored based on the photograph obtained by the imaging unit 3, thereby improving performance even for large temperature changes and large microparticles.

[0045] Although the method for controlling the timing at which the droplets break off is not particularly limited, it is preferable to adjust the voltage of the vibration element 13 based on the timing at which the droplets break off identified from the multiple photographs.

[0046] The control method by the control unit 3 will be described more specifically based on the following control examples 1 to 4.

[0047] [Control example 1] In this control example 1, first, droplets containing microparticles are automatically adjusted to create droplets that do not splash (see A in FIG. 5). After that, the strobe is swung to obtain continuous photographs of phases within a certain cycle, and in the continuous photographs, the phase at which the droplets break off (the phase at which the droplets break off) is identified (see B in FIG. 5). Specifically, for example, the light emission cycle of the strobe is divided into equal parts (e.g., 16 parts), and the phase at which the droplets break off is detected from the images of the continuous photographs in one cycle. After that, continuous photographs that are further divided into phases before and after this phase are obtained, and the phase is determined. The phase of the timing at which the droplet breaks off in the divided continuous photographs is more accurately identified (see C in FIG. 5). Specifically, for example, the light emission cycle of the strobe is re-divided into equal parts (for example, 40 parts) to more accurately detect the phase at which the droplet breaks off from the images of one cycle of continuous photographs.

[0048] In addition, in this control example 1, before acquiring a series of photographs further divided into phases before and after the phase in which the droplets break off, it may be confirmed that the droplets are also in a broken-off state in the phase next to the phase in which the droplets break off.

[0049] FIG. 6 is a drawing substitute photograph showing a series of photographs in which one cycle is divided into 16 equal parts. This drawing substitute photograph is a series of phase photographs obtained by changing the strobe emission start time of the strobe provided in the imaging unit 3. The term "one cycle" here means the time it takes to form one droplet containing a microparticle, and is determined by the frequency of the vibration element 13 (e.g., a piezoelectric element). The vibration element 13 frequency is about 10 kHz to 50 kHz, and the time of one cycle is about 1 / 10000 seconds at maximum and about 1 / 50000 seconds at minimum.

[0050] Then, the position of break-off is monitored to check how many times the droplets break off and how many times they do not break off within a certain period of time (for example, 10 seconds). Thereafter, if the number of breaks exceeds a threshold value (for example, 40 to 60%, preferably 50%), the droplets are biased toward breaking off, so the voltage of the vibration element 13 is lowered to make them less likely to break off. On the other hand, if it is below the threshold value, the droplets are biased toward sticking together, so the voltage of the vibration element 13 is increased to make them more likely to break off. That is, after acquiring images by dividing one cycle into certain phases, the phase at which breakage occurs is identified, and the voltage of the vibration element 13 is adjusted so that the number of breaks at that phase is, for example, 40 to 60%, preferably 50%.

[0051] [Control example 2] In this control example 2, similarly to control example 1, first, droplets containing microparticles are automatically adjusted to create droplets that do not splash (see A in FIG. 7). After that, a strobe is swung to obtain continuous photographs of phases within a certain period, and the phase at which the droplet breaks off (the phase at which the droplet breaks off) is identified in the continuous photographs (see B in FIG. 7). In this identification, the phase at which the droplet breaks off is not identified as precisely as in control example 1 described above, but if the distance of the gap caused by the breakage is sufficiently small, control similar to control example 1 is possible.

[0052] Then, the distance of the gap that occurs when the droplet breaks off is monitored, the distance (=Gap) between the break-off point of the phase at which the identified droplet breaks off and the satellite is measured, and the voltage of the vibration element 13 is adjusted so that the droplet breaks off at the same phase and the gap length can be maintained constant. If the gap becomes larger, the droplet is biased toward breaking off, so the voltage of the vibration element 13 is lowered to make the gap smaller. On the other hand, if the gap becomes smaller, the droplet is biased toward sticking together, so the voltage of the vibration element 13 is increased to make it easier to break off.

[0053] [Control example 3] Control example 3 is an example of control when a fast satellite occurs. In Example 3, first, a droplet containing a microparticle is created (the droplet may be wet at this point) (see A in FIG. 8). After that, the voltage of the vibration element 13 is adjusted so that the phase of the timing at which the droplet breaks off corresponds to the opposite direction of the phase at which the microparticle is charged within a certain period (see B in FIG. 8).

[0054] When a fast satellite occurs, the previous droplet breaks off and the charge If the order of the start of the jet, the breakage of the target droplet, and the end of the charge is not observed, splashes will occur. In other words, since the droplets separate as satellites after being torn off from the liquid column, it is only necessary to pay attention to the droplets being torn off, and control is possible using this method. As a specific droplet control method, the methods described in the above-mentioned Control Example 1 and Control Example 2 can be used.

[0055] In addition, in this control example 3, before the voltage control by the vibration element 13, a strobe may be swung to obtain a plurality of photographs of the phase at a certain time.

[0056] [Control example 4] Control example 4 is an example of control when a slow satellite occurs. In Example 4, first, droplets containing microparticles are created (the droplets may be wet at this point) (see A in FIG. 9). After that, a strobe is swung to obtain multiple phase photographs at a certain time, and the manner in which the satellites of the droplets break off is examined to identify the phase in which the droplet in front of the droplet (the droplet containing the target microparticle) breaks off from the liquid column and the phase in which the satellite of the droplet breaks off from the liquid column (see B and C in FIG. 9). Then, the phase difference in the break-off of these satellites is examined, and the voltage of the vibration element 13 is adjusted so that, for example, the phase in which the electric charge is charged is located in the middle.

[0057] When a slow satellite occurs, droplets containing the target microparticles If the order of events is not observed, splashing will occur. In the case of control example 3 (Fast satellite), the droplet breaks off from the liquid column only once, so it is sufficient to adjust the voltage of the vibration element 13 so that the phase in which the droplet breaks off corresponds to the opposite direction to the phase in which the microparticle is charged within one period. However, in the case of Slow Satellite, if the phase is adjusted to the opposite direction, the droplet before the droplet containing the target microparticle breaks off from the liquid column, the satellite of the droplet breaks off from the liquid column, and charging begins. This results in the wrong order, causing splashing. Therefore, to avoid this, it is necessary to identify the phase in which the droplet in front of the droplet (the droplet containing the target microparticle) breaks off from the liquid column and the phase in which the satellite of the droplet breaks off from the liquid column, measure the phase difference of the satellite breakoff by shaking a strobe, etc., and adjust so that the charge phase is in the middle. As a specific droplet control method, the methods described in the above-mentioned Control Example 1 and Control Example 2 can be adopted.

[0058] In the present technology, the control unit 4 may be configured to be able to control each unit of the microparticle analysis device 1 according to the present technology. Also, the control unit 4 may be connected to each unit via a network.

[0059] (6) Detection unit 5 Reference numeral 5 in Fig. 1 denotes a detection unit 5 that detects measurement target light generated from microparticles such as cells by irradiation with a laser L1 emitted from a light source 51. The detection unit 5 detects microparticles in a fluid flowing through the flow channel. The detection unit 5 detects characteristics of cells between the narrowing section 261 and the narrowing section 262 of the sample flow channel 22. The detection of the characteristics is not particularly limited, but for example, in the case of optical detection, the scattered light and fluorescence generated from the cells are detected by the detection unit 5 by irradiating the cells arranged in a row at the center of the three-dimensional laminar flow through the sample flow channel 22 with a laser L1 (see Fig. 1).

[0060] In this light irradiation and detection, in addition to the laser light source, an irradiation system such as a condenser lens, a dichroic mirror, and a band pass filter for condensing and irradiating the laser on the cell may be configured. The detection system may be, for example, a PMT (photomultiplier tube) or a C It is composed of area imaging elements such as CD and CMOS elements.

[0061] The light to be measured detected by the detection system of the detection unit 5 is light generated from the cells by irradiation with the measurement light. Specifically, for example, it is forward scattered light, side scattered light, scattered light such as Rayleigh scattering and Mie scattering, etc. This light to be measured is converted into an electric signal, output to the control unit 4 described above, and used to determine the optical properties of the cells.

[0062] The detection unit 5 may detect the characteristics of cells magnetically or electrically. In this case, microelectrodes are disposed facing each other in the sample flow channel 22 of the microchip 2, and resistance, capacitance, inductance, impedance, change in the electric field between the electrodes, magnetization, magnetic field change, magnetic field change, etc. can be measured.

[0063] (7) Deflection plates 61, 62 Reference numerals 61 and 62 in Fig. 1 indicate a pair of deflection plates arranged opposite to each other with a droplet ejected from the orifice 21 and imaged by the imaging unit 3 therebetween. The deflection plates 61 and 62 are configured to include electrodes that control the movement direction of the droplet ejected from the orifice 21 by an electrical force acting on the droplet with an electric charge. The deflection plates 61 and 62 also control the trajectory of the droplet generated from the orifice 21 by an electrical force acting on the droplet with an electric charge. In Fig. 1, the opposing direction of the polarizing plates 61 and 62 is indicated by the X-axis direction.

[0064] (8) Collection containers 71-73 In the microparticle analysis device 1 according to the present technology, droplets are received in one of a plurality of collection containers 71-73 arranged in a row in the opposing direction of the deflection plates 61, 62 (X-axis direction). The collection containers 71-73 may be general-purpose plastic tubes or glass tubes for experimental use. There is no particular limit to the number of collection containers 71-73, but here, a case in which three are installed is illustrated. The droplets generated from the orifice 21 are guided and collected into one of the three collection containers 71-73 depending on the presence or absence of an electrical acting force between the deflection plates 61, 62 and the magnitude of the force.

[0065] The collection vessels 71 to 73 may be replaceably installed in a container for collection vessels (not shown). The container for collection vessels is disposed on a Z-axis stage (not shown) configured to be movable in a direction (Z-axis direction) perpendicular to the discharge direction of droplets from the orifice 21 (Y-axis direction) and the opposing direction of the polarizing plates 61 and 62 (X-axis direction).

[0066] (9) Analysis section 8 The analysis unit 8 is connected to the detection unit 5 and analyzes the detection value of the light for the microparticles detected by the detection unit 5.

[0067] For example, the analysis unit 8 can correct the detection value of the light received from the detection unit 5 and calculate the feature amount of each microparticle. Specifically, feature amounts indicating the size, shape, internal structure, etc. of the microparticle are calculated from the detection values ​​of the received fluorescence, forward scattered light, and back scattered light. In addition, a separation decision can be made based on the calculated feature amounts and the separation conditions, etc. received in advance from the input unit, and a separation control signal can be generated.

[0068] The analysis unit 8 is not essential for the particle analyzer 1 according to the present technology, and it is also possible to analyze the state of the microparticles using an external analysis device or the like based on the detection value of the light detected by the detection unit 5. In addition, the analysis unit 8 may be connected to each unit via a network.

[0069] (10) Storage section 9 In the memory unit 9, the values ​​detected by the detection unit 5, the feature amounts calculated by the analysis unit 8, the fractionation control signals, It stores all items related to the measurement, such as the number and the separation conditions inputted through the input section.

[0070] In the microparticle analysis device 1, the storage unit 9 is not essential, and an external storage device may be connected. For example, a hard disk or the like may be used as the storage unit 9. In addition, the recording unit 9 may be connected to each unit via a network.

[0071] (11) Display section 10 The display unit 10 can display all items related to the analysis, such as values ​​detected by the detection unit 5. Preferably, the display unit 10 can display the feature amount for each microparticle calculated by the analysis unit 8 as a scattergram.

[0072] In the microparticle analysis device 1 according to the present technology, the display unit 10 is not essential, and an external display device may be connected. As the display unit 10, for example, a display, a printer, etc. may be used.

[0073] (12) Input section The input unit (not shown) is a portion for a user such as an operator to operate. The user can access the control unit 3 through the input unit and control each part of the microparticle analysis device 1 according to the present technology. Preferably, the input unit can set an area of ​​interest for the scattergram displayed on the display unit 10 and determine the sorting conditions.

[0074] In the microparticle analysis device 1 according to the present technology, the input unit is not essential, and an external operation device may be connected. As the input unit, for example, a mouse, a keyboard, etc. may be used.

[0075] (13)Other In addition, in the present technology, the functions performed by each part of the microparticle analysis device 1 according to the present technology can be stored as a program in hardware resources including a personal computer, a control unit including a CPU, etc., and a recording medium (e.g., a non-volatile memory (e.g., a USB memory, etc.), a HDD, a CD, etc.), and can be operated by the personal computer or the control unit.

[0076] 2. Microparticle analysis method In the microparticle analysis method according to the present technology, at least a droplet forming step, a charge charging step, an imaging step, and a control step are performed. In addition, other steps may be performed as necessary. Each step will be described in detail below.

[0077] (1) Droplet formation step In the droplet formation step, a fluid consisting of a sample flow containing microparticles and a sheath flow flowing so as to enclose the sample flow is vibrated by a vibration element to form droplets in the fluid. The specific method performed in the droplet formation step is similar to the method performed in the droplet formation unit described above, and therefore a description thereof will be omitted here.

[0078] (2) Charge step In the charge charging step, the liquid containing the microparticles is charged with an electric charge. The specific method of the charge charging step is the same as the method performed in the charge charging unit 11 described above, and therefore a description thereof will be omitted here.

[0079] (3) Imaging step In the imaging step, a photograph of a phase at a certain time is obtained. The specific method performed in the imaging step is similar to the method performed by the imaging unit 3 described above, and therefore a description thereof will be omitted here.

[0080] (4) Control step The specific method performed in the control step is similar to the method performed by the control unit 4 described above, and therefore a description thereof will be omitted here.

[0081] In addition, in the control step, it is preferable to adjust the voltage of the vibration element 13 based on the timing at which the droplet breaks off, which is specified by the multiple photographs.

[0082] [An example of the flow of microparticle analysis] An example of a flow of microparticle analysis using the microparticle analysis method according to the present technology will be described with reference to Fig. 10. Note that the processing of each step in the flowchart shown in Fig. 10 is performed by, for example, each of the above-mentioned units.

[0083] First, the sheath liquid is discharged from the orifice 21 of the microchip 2 (step S1). After that, the control unit 4 changes the frequency and voltage of the piezoelectric element, which is the vibration element 13 (step S2). Then, the control unit 4 judges the droplet shape (step S3). If the droplet shape is poor, the process returns to step S2. If the droplet shape is good, other adjustments are made (step S4). In step S4, specifically, an adjustment is made such as swinging a strobe to find a break-off point, but this is not an essential step in the microparticle analysis method according to the present technology. Steps S1 to S4 are steps for automatically adjusting the droplet, and by performing these steps, the device is provided in advance with droplet conditions suitable for separation (for example, the length and position of the droplet, the direction in which the satellite is absorbed (Fast or Slow), etc.), and the frequency and voltage of the vibration element 13 are changed to search for droplets that meet the desired conditions.

[0084] After step S4, the control unit 4 starts droplet control (step S5). First, the control unit 4 detects a monitoring point (step S6). The monitoring point is a moment when a droplet containing a microparticle breaks off at a phase at a certain time. To target this monitoring point, the methods described in the above-mentioned control examples 1 to 4 can be mentioned. A judgment is made about a set threshold value for this monitoring point (step S7), and if the value of the monitoring point is larger than the threshold value, the voltage of the vibration element 13 is lowered (step S8). On the other hand, if the value of the monitoring point is smaller than the threshold value, the voltage of the vibration element 13 is raised (step S9). Steps S4 to S9 are steps for controlling droplets. By performing these steps, the part where the droplet breaks off from the liquid column is continuously monitored, and this is directly maintained to keep the timing of break-off constant, thereby realizing stable sorting.

[0085] In addition, the present technology can also have the following configuration. (1) a flow path through which a fluid including a sample flow containing microparticles and a sheath flow flowing so as to enclose the sample flow; a droplet forming unit that applies vibration to the fluid using a vibration element to form droplets in the fluid; a charge charging unit that charges droplets containing the microparticles; An imaging unit for obtaining a phase image at a certain time; A control unit that controls the timing at which the droplets break off based on the photograph; A microparticle analysis device comprising at least the above. (2) The microparticle analysis device according to (1), wherein the control unit adjusts the voltage of the vibration element based on the timing at which the droplets break off identified by the multiple photographs. (3) The control unit acquires continuous photographs of phases in one period, and identifies a phase at which the droplet breaks off in the continuous photographs. Child analyzer. (4) The microparticle analysis device according to (3), wherein the control unit acquires a series of photographs that are further divided into phases before and after the phase of the timing at which the droplets break off, and identifies the phase of the timing at which the droplets break off in the divided series of photographs. (5) the imaging unit includes a strobe; The microparticle analysis device according to (3) or (4), wherein the identification is performed using continuous phase photographs obtained by changing a strobe emission start time of the strobe. (6) The microparticle analysis device according to any one of (1) to (5), wherein the control unit controls the phase of the timing at which the droplet breaks off to be a phase corresponding to an opposite direction to the phase at which the droplet is charged. (7) The microparticle analysis device according to any one of (1) to (6), wherein the control unit controls the phase in which the electric charge is charged to the droplet to be between the phase in which a droplet preceding the droplet breaks off from the liquid column and the phase in which a satellite of the droplet breaks off from the liquid column. (8) a droplet forming step of applying vibration to a fluid including a sample flow containing microparticles and a sheath flow flowing so as to enclose the sample flow, using a vibration element, to form droplets in the fluid; a charge charging step of charging droplets containing the microparticles with an electric charge; an imaging step for obtaining a phase picture at a time; a control step of controlling a timing at which the droplets break off based on the photograph; The method for analyzing microparticles comprises at least the steps of: (9) The microparticle analysis method according to (8), wherein in the control step, the voltage of the vibration element is adjusted based on the timing at which the droplet breaks off identified by the multiple photographs. [Explanation of symbols]

[0086] 1: Microparticle analyzer 11: Electric charge section 12: Electrode 13: Vibration element 2: Microchip 2a, 2b: Substrate layer 21: Orifice 22: Sample flow path 23: Sample inlet 24: Seas Inlet 25: Suction channel 27: Straight section 211: Notch 251: Suction outlet 252: Connecting port 261, 262: Narrowing section 3: Imaging section 31: Strobe 32: Droplet Camera 4: Control unit 5: Detection section 51:Light source 61, 62: Deflection plate 71~73: Collection container 8: Analysis section 9: Storage part 10: Display section

Claims

1. a vibration element for imparting vibrations to the fluid discharged from the orifice to form droplets containing microparticles; an imaging unit that acquires a plurality of images of the flow of the fluid over a predetermined period of time; a control unit that identifies a phase at which the droplet breaks off during the predetermined period based on the plurality of images, and adjusts a voltage of the vibration element based on a position at which the droplet breaks off in an image acquired at the identified phase; A microparticle analysis device comprising:

2. The imaging unit acquires a plurality of additional images obtained by further dividing a phase before and after the phase in which the droplet breaks off during the predetermined period, The control unit identifies a second phase during the predetermined period in which the droplet breaks off based on the plurality of additional images; and The microparticle analysis device according to claim 1 , wherein the adjustment to the voltage of the vibration element is made based on a position where the droplet breaks off in an image acquired at the identified second phase.

3. The control unit identifies at least one of the number of times the droplet breaks off and the number of times the droplet does not break off in the image acquired at the determined second phase, The microparticle analysis device according to claim 2 , wherein the adjustment to the voltage of the vibration element is performed based on at least one of the identified number of times the droplet breaks off and the identified number of times the droplet does not break off.

4. The control unit determines a distance from a position where the droplet breaks off to a satellite in an image acquired at the determined phase, 2. The particle analyzer of claim 1, wherein the adjustment to the voltage of the transducer element is based on a distance from the location where the droplet breaks off to a satellite.

5. the imaging unit includes a strobe; The microparticle analysis device according to claim 1 , wherein the phase is specified using images acquired by changing a strobe emission start time of the strobe.

6. The microparticle analysis device according to claim 1 , further comprising a charge charging section configured to charge droplets containing the microparticles.

7. The microparticle analysis device according to claim 6 , wherein the adjustment to the voltage of the vibration element is performed so that the specified phase corresponds to a phase that corresponds to an opposite direction to a phase for charging the droplet.

8. The microparticle analysis device according to claim 6, wherein the adjustment to the voltage of the vibration element is performed so that the phase in which the droplet is charged is between the phase in which a droplet preceding the droplet breaks off from the liquid column and the phase in which a satellite of the droplet breaks off from the liquid column.

9. The microparticle analysis device according to claim 1 , wherein the fluid includes a sample flow containing microparticles and a sheath flow containing the sample flow.

10. The microparticle analysis device according to claim 1 , wherein the microparticle is a cell.

11. Further comprising a microchip having the orifice. The microparticle analysis device according to claim 1 .

12. a droplet forming step of applying vibration to the fluid discharged from the orifice using a vibration element to form droplets containing microparticles; an imaging step of acquiring a plurality of images of the flow of the fluid over a predetermined period of time; a control step of identifying a phase at which the droplet breaks off during the predetermined period based on the plurality of images, and adjusting a voltage of the vibration element based on a position at which the droplet breaks off in an image acquired at the identified phase; A method for analyzing microparticles.

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