Particle sorting method, particle sorting system, and program

Abstract

The present invention provides a technique capable of efficiently sorting particles even if the particles to be sorted are large.　Provided is, for example, a particle sorting method including: a step for supplying a first drive voltage to a vibrating element that imparts vibration to a fluid; a step for acquiring first optical information of the fluid and droplets generated by the vibration of the vibrating element by the first drive voltage; a step for supplying a second drive voltage lower than the first drive voltage to the vibrating element; a step for acquiring second optical information of the fluid and droplets, containing second particles, generated by the vibration of the vibrating element by the second drive voltage; and a step for determining a parameter for sorting the second particles on the basis of a comparison between the first optical information and the second optical information.

Description

Particle sorting method, particle sorting system, and program

[0001] The present technology relates to a particle sorting method, a particle sorting system, and a program.

[0002] Flow cytometry has been used as a method for analyzing (or analyzing; in this technology, analysis includes the concept of analyzing) or separating particles such as cells, microorganisms, and ribosomes. An apparatus used for flow cytometry is called a flow cytometer (FCM). Among flow cytometers, an apparatus used for separating (also called "sorting") particles is particularly called a cell sorter.

[0003] In a flow cytometer, particles flowing through a flow channel are irradiated with laser light of a specific wavelength, and the light emitted from each particle, such as fluorescence, forward scattered light, and side scattered light, is converted into an electrical signal by a detector and digitized, and the results are subjected to statistical analysis to determine the type, size, structure, etc. of each individual microparticle. In addition, particles to be sorted can be sorted by applying an electric charge to them using a vibration element, controlling their trajectory with a deflection plate, and guiding them to the desired collection container.

[0004] It is known that the yield of large particles decreases when separating large particles. The reasons for this include droplet turbulence and charge inhibition by particles located between droplets. In response to this, Patent Documents 1 and 2 disclose techniques for extending the section in which particles are charged (the so-called charging section) when the particles to be separated are large.

[0005] JP 2019-063798 A JP 2015-152439 A

[0006] However, in the technology for separating large-diameter particles, there is no generally known method for improving the state in which the droplet shape is disturbed during particle delivery, resulting in a decrease in separation performance.

[0007] Therefore, the main object of the present technology is to provide a technology that can efficiently separate particles even when the particles to be separated are large.

[0008] The present technology provides a particle sorting method including the steps of first supplying a first drive voltage to a vibration element that applies vibration to a fluid, acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element at the first drive voltage, supplying a second drive voltage lower than the first drive voltage to the vibration element, acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element at the second drive voltage, and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0009] The present technology also provides a particle sorting system including: a voltage supply unit that supplies a first drive voltage to a vibration element that imparts vibrations to a fluid; and an imaging unit that acquires first optical information of the fluid and droplets generated by the vibration of the vibration element with the first drive voltage, wherein the voltage supply unit supplies a second drive voltage lower than the first drive voltage to the vibration element; the imaging unit acquires second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element with the second drive voltage; and a control unit that determines parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0010] The present technology also provides a program that executes the following steps: supplying a first drive voltage to a vibration element that imparts vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element with the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element with the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0011] In this specification, the term "large-diameter particles" refers to particles having a particle diameter exceeding approximately 1 / 5 of the orifice diameter, which is generally the particle size at which stable sorting can be performed in a cell sorter, and specifically refers to particles having a particle diameter of, for example, approximately 10 μm to approximately 100 μm.

[0012] 1 is a schematic diagram showing the relationship between the combination of waves caused by vibration element V and waves caused by large-diameter particles, and the droplet generation position. FIG. 2 is a diagram showing an example of the overall flow of a particle sorting method according to the present technology. FIG. 3 is a diagram showing an example of a measurement flow for BOT_1. FIG. 4 is a diagram showing an example of a measurement flow for BOT_2. FIG. 5 is a diagram showing an example of a setup flow for BOT_1. FIG. 6 is a diagram showing an example of a setup flow for BOT_2. FIG. 7 is a diagram showing an example of a measurement flow for BOP_2. FIG. 8 is a diagram showing an image of BOT_1. FIG. 9 is a diagram showing an image of BOT_2. FIG. 10 is a diagram showing an image of BOP_1. FIG. 11 is a diagram showing an image of BOP_2. FIG. 12 is a schematic conceptual diagram showing a first embodiment of a particle sorting system 1 according to the present technology. FIG. 13 is a schematic conceptual diagram showing a second embodiment of a particle sorting system 1 according to the present technology. FIG. 14 is a diagram showing an example of an image of a liquid column containing particles at each elapsed time (clock unit). FIG. 15 is a diagram showing elapsed time (Delay) vs. droplet generation rate (M / N) vs. recovery rate (Recovery). FIG. 16 is a diagram showing an example of a method for determining before and after droplet generation. 27 is a diagram showing an example of an image of one particle (one strobe) and an image of multiple particles (multiple strobes). FIG. 28 is a diagram showing an example of image selection based on the average brightness value of each image. FIG. 29 is a diagram showing the recovery rate (Recovery) vs. ΔBOT of 30 μm Beads. FIG. 30 is a diagram showing an image of a charged region that may be separated together with particle-induced droplets. FIG. 31 is a diagram explaining the concept of multi-drop. FIG. 32 is a diagram explaining Example 3. FIG. 33 is a diagram explaining Example 3. FIG. 34 is a diagram explaining Example 3. FIG. 35 is a diagram explaining Example 3. FIG. 36 is a diagram explaining Example 3. FIG. 37 is a diagram explaining Example 3. FIG. 38 is a diagram explaining Example 3. FIG. 39 is a diagram explaining Example 4. FIG. 40 is a flowchart showing a method for setting a charge section in the case shown in FIG. 26. FIG. 41 is a diagram explaining Example 4. FIG. 42 is a flowchart showing a method for setting a charge section in the case shown in FIG. 28. FIG. 43 is a diagram showing the results when second particles are separated using multi-drop.

[0013] Preferred embodiments for implementing the present technology will be described below with reference to the drawings. The embodiments described below are examples of typical embodiments of the present technology, and are not intended to narrow the scope of the present technology. The description will be given in the following order: 1. First Embodiment (Particle Sorting Method) (1) Background Art (2) Overview of the Present Technology (3) Details of the Particle Sorting Method According to the Present Embodiment <Adjustment Using ΔBOT (Break-Off Time)> <Adjustment Using ΔBOP (Break-Off Point)> 2. Second Embodiment (Particle Sorting System 1) (1) Flow Channel P (2) Light Irradiation Unit 11 (3) Light Detection Unit 12 (4) Vibration Element V (5) Voltage Supply Unit 13 (6) Sorting Unit 14 (7) Optical Information Acquisition Unit 15 (8) Control Unit 16 (9) Analysis Unit 17 (10) Storage Unit 18 (11) Display Unit 19 (12) Others 3. Third Embodiment (Program)

[0014] 1. First embodiment (particle sorting method)

[0015] (1) Background technology

[0016] As mentioned above, the particle size that a cell sorter can handle is generally limited to approximately 1 / 5 of the orifice diameter, and sorting particles larger than this size can result in a decrease in yield. The reasons for this include irregular droplet shapes and charge inhibition caused by particles located between droplets.

[0017] However, there is no generally known method for improving the condition in which droplet shape becomes distorted during particle delivery, resulting in reduced separation performance. Furthermore, the degree of separation performance degradation varies depending on various conditions and states of the device and particles. Even if there are parameter adjustments that can improve the recovery rate, the effectiveness of such adjustments cannot be confirmed without actually performing separation and checking the recovery rate.

[0018] One way to improve the separation performance is to increase the recovery rate of particles located at the center of the droplet. However, although it is possible to select and sort particles located at the center of the droplet, there is no way to ensure that all particles are located at the center of the droplet, and particles located at the edge of the droplet will be discarded. This improves the recovery rate, but inevitably reduces the yield.

[0019] (2) Overview of this technology

[0020] The impetus for this technology came from the discovery that, by observing the liquid column containing the large particles (the position the particles reach) in relation to the elapsed time from particle detection by laser during the delivery of large particles, waves and droplets appear in the liquid column as the particles move forward, a phenomenon independent of the control of the drive voltage.

[0021] FIG. 1 is a schematic diagram showing the relationship between the wave generated by the vibration element V and the wave generated by the large particle, and the droplet generation position. The left diagram in FIG. 1 shows the droplet generation position when the vibration element is turned on / off. The middle diagram in FIG. 1 shows the droplet generation position when the vibration element is turned off and the particle size is changed. The right diagram in FIG. 1 schematically shows the droplet generation position in the combined wave state when the vibration element is turned on and the particle size is changed. That is, the left diagram in FIG. 1 shows the droplet generation position by controlling the drive voltage, and the middle diagram in FIG. 1 shows the droplet generation position due to the wave generated by the large particle when the particle size of the large particle is changed, and the right diagram in FIG. 1 shows the droplet generation position in the combined wave state of the left diagram in FIG. 1 and the middle diagram in FIG. 1 (specifically, the combined waves are b = a + b', c = a + c', and d = a + d', respectively). 1 shows the state of the BOP formed by the vibration element in the absence of particles (the BOP varies depending on the machine), while FIG. 1 shows the state where droplets are predominantly broken up by the vibration element V, while FIG. 1 shows the state where the combining state is diverse and difficult to control, and FIG. 1 shows the state where tearing due to waves of large particles is predominant. The tearing stability here is b>d>>c.

[0022] In Figure 1, droplets induced by controlling the drive voltage are known, but the waves and droplets induced by the progression of large-diameter particles are generally unknown. Therefore, the state of the two waves combined when large-diameter particles are fed is also unknown, and the droplet generation position of the former (droplet generation position of droplets induced by controlling the drive voltage; see a in Figure 1) varies from device to device, and the droplet generation position of the latter (progression of large-diameter particles to droplet wave; see b' to d' in Figure 1) varies depending on the particle size, resulting in various aspects of performance degradation.

[0023] Furthermore, while the prior art documents deal with large particles in a uniform manner by extending the charging interval, the present technology not only extends the charging interval but also calculates ΔBOT or ΔBOP to determine the possibility of performance degradation and improves performance degradation by determining additional parameters.

[0024] That is, this technology is primarily intended to facilitate efficient separation of large-diameter particles based on the combining state of the two waves, and is a method for automatically determining separation performance and setting up for performance improvement as necessary. This allows for prediction of possible degradation of separation performance without performing separation, thereby saving time and effort and also improving the separation performance itself. Furthermore, since degradation of separation performance can be predicted without performing separation, sample waste can be prevented.

[0025] Specifically, the outline of this technology is to separately calculate the position of droplets formed by amplitude control of the vibration element V or by large-diameter particles, and the time elapsed from the detection of the particles until they reach that position. For the two types of droplet generation described above, the difference between the positions at which droplets are generated and torn off, i.e., the break-off points (also referred to as "BOP"), is defined as ΔBOP. Also, the difference between the times at which droplets are generated, i.e., the break-off times (also referred to as "BOT"), is defined as ΔBOT.

[0026] On the other hand, the detection laser position and the "detection time" of the particle at the detection laser position are considered as the reference. The part of the liquid column ejected from the orifice containing the particle is observed over time, and images of the waves (and droplets) appearing in the liquid column as the particle moves forward are acquired. Based on the acquired images, the position of droplet generation or the elapsed time until droplet generation is calculated (however, particles are not necessarily required for the calculation of BOP_1, which will be described later). Up to this point, the drive of the vibration element V is OFF or driven at low output.

[0027] This technology can predict a decline in sorting performance based on the ΔBOT or ΔBOP value. If a decline in sorting performance is expected, additional adjustments to droplet control are made. Specifically, if the two BOPs or BOTs are similar, or if the latter BOP is longer, or if the BOT is slower, the drive output of the vibration element V is reduced and adjustments are made to target the droplets for sorting. When sorting droplets, droplets are generated in the preceding liquid column, and these liquid columns and droplets also break off within the charge section, so there is a possibility that they will be sorted. Therefore, a function may be provided to prevent sorting if particles not to be sorted exist between droplets torn by the piezoelectric element and those torn by particles, or within the droplets. Furthermore, since it is preferable to minimize the area where particles not to be sorted exist, the temporal or positional distance between droplets torn by the piezoelectric element and those torn by particles may be adjusted. In this case, the break-off timing of droplets torn by the piezoelectric element may be controlled to be constant.

[0028] (3) Details of the particle sorting method according to this embodiment

[0029] The particle sorting method according to this embodiment comprises at least the steps of: supplying a first drive voltage to a vibration element V that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element V with the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element V; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element V with the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0030] The cause of the degradation of the separation performance of large-diameter particles (hereinafter also referred to as "second particles") is a phenomenon in which droplets, which are controlled to break off at a fixed timing by driving the vibration element V, become unstable due to the flow of large-diameter particles. This technology has discovered that this unstable phenomenon can be decomposed into two phenomena: a wave caused by the drive control of the vibration element V and a wave generated independently as the second particles advance. This technology predicts whether separation performance will be degraded by observing each wave and the resulting droplet break-off, and also sets up an apparatus to improve performance.

[0031] In the particle sorting method according to this embodiment, the setup of the device may be performed based on either ΔBOT (break-off time) or ΔBOP (break-off point).

[0032] The particle sorting method according to this embodiment will be described in detail below.

[0033] <Adjustment using ΔBOT (break off time)>

[0034] Below, a method for calculating ΔBOT and predicting the deterioration of preparative separation performance will also be explained.

[0035] ΔBOT=BOT_2 (hereinafter also referred to as "second BOT")-BOT_1 (hereinafter also referred to as "first BOT"), and can be determined by measuring BOT_1 and BOT_2.

[0036] 2 is a diagram showing an example of the overall flow of the particle sorting method according to the present technology. In this embodiment, first, BOT_1 and BOT_2 are measured (S101, S102). Next, ΔBOT is calculated, and if ΔBOT is equal to or less than a threshold (S103), setup for BOT_2 is performed (S104). If ΔBOT exceeds the threshold, setup for BOT_1 is performed (S105).

[0037] Each step will be described in detail below.

[0038] [Step of supplying a first driving voltage to the vibration element V that applies vibration to the fluid]

[0039] FIG. 3 is a diagram showing an example of the measurement flow of BOT_1. FIG. 8 is a diagram showing an image of BOT_1. Here, the vibration element V vibrates the flow path at a predetermined frequency to impart minute vibrations to the sheath liquid, which breaks the fluid (sample liquid and sheath liquid) discharged from the orifice into droplets, generating a flow of droplets. The vibration element V is not particularly limited, and can be freely selected and used as appropriate depending on the purpose. Specifically, for example, a piezoelectric vibration element V or the like can be used.

[0040] A first driving voltage is supplied to a vibration element V that vibrates the fluid, thereby vibrating the fluid and forming a first droplet suitable for collection (S201). In other words, by controlling the vibration element V, a droplet suitable for collection and having the shortest BOP is formed. Calibration beads can be used to measure BOT_1.

[0041] [Step of acquiring first optical information of fluid and droplets generated by vibration of the vibration element V due to the first drive voltage]

[0042] In this step, first optical information of the fluid and droplets generated by the vibrations applied in the above-described step is acquired, for example, by the optical information acquisition unit 15 described below. The optical information may be, for example, at least one of an image and fluorescent information (e.g., fluorescent information of a side stream).

[0043] This process also includes a step of delivering calibration beads (hereinafter also referred to as "first particles") (S203), detecting the particles by scattered light or fluorescence (S204), and determining the time from the reference time until the droplet containing the first particles breaks off as a first BOT (BOT_1) based on the first optical information. In other words, BOT_1 is the time from the detection of a particle in a droplet suitable for sorting to the breakoff of the droplet containing the particle by driving and controlling the vibration element V. This can be obtained by calculating the elapsed clock at which the first particle arrives at the droplet generating unit (S205).

[0044] In this embodiment, the timing of particle detection is defined as the "reference time" in a particle sorting system 1 (described later) that is configured to detect scattered light or fluorescence generated by irradiating light on flowing particles. The particle sorting system 1 (described later) has an optical information acquisition unit 15 that observes the liquid column that is movable in the direction of the liquid column's movement, and has an LD or LED on the opposite side that can emit light at any timing and for any emission time.

[0045] [Step of supplying a second driving voltage lower than the first driving voltage to the vibration element V]

[0046] FIG. 4 is a diagram showing an example of the measurement flow of BOT_2. FIG. 9 is a diagram showing an image of BOT_2. In this step, a second drive voltage lower than the first drive voltage (including the case where the drive voltage is turned off) is supplied to the vibration element V (S301). The drive voltage is, for example, 1% to 20%, preferably 5% to 15%, of the drive voltage during normal droplet control drive.

[0047] In this embodiment, the first optical information is optical information about first particles (calibration beads) that are different in size from the second particles, and it is assumed that the second particles have a larger particle size than the first particles.

[0048] In this process, a sample liquid containing large-diameter particles is delivered (S302), and a second driving voltage is supplied to the vibration element V that vibrates the fluid, thereby vibrating the fluid and forming waves caused by the second particles (large-diameter particles).

[0049] [Step of acquiring second optical information of fluid and droplets containing second particles generated by vibration of the vibration element V due to the second driving voltage]

[0050] In this step, second optical information of the fluid and droplets generated by the vibrations applied in the above-described step is acquired, for example, by the optical information acquisition unit 15 (described later) (S303). The optical information may be, for example, at least one of an image and fluorescent information (e.g., fluorescent information of a side stream). The droplets included in the second optical information are formed by waves generated by second particles (large-diameter particles).

[0051] The time until the droplet containing the second particle breaks off is designated BOT_2. In other words, BOT_2 is the time from the detection of the particle to the break-off of the droplet containing the particle, in droplets induced by the particle's progression.

[0052] A specific method for measuring BOT_2 involves observing the liquid column containing the second particle (S304). Note that in S304, n is any of X, 1, and Y, and X and Y are any natural numbers. Next, the position where the particle reaches after n clocks have elapsed since particle detection is photographed and observed using the imaging unit 151 (described later) (S305). Next, α images are acquired after n clocks have elapsed since particle detection (S306). Next, out-of-focus images (β images) are removed, and images of droplet generation are counted (γ images) (S307). Next, the liquid column containing the particle is observed (S308).

[0053] This process further includes a step of calculating the occurrence rate of droplets containing the second particles based on the second optical information. Specifically, as described above, optical information (here, image acquisition) of the liquid column containing the second particles is acquired according to the elapsed time from the reference time. The interval of the elapsed time is, for example, one clock unit of the piezo drive. To acquire the optical information (preferably, the image), the LED is strobed at the moment of the elapsed time. The imaging unit 151 is moved to a position where the arrival position of the particle can be observed at each elapsed time, and multiple images are acquired at each elapsed time, and the occurrence rate of droplets is calculated. In other words, here, the occurrence rate of droplets is calculated from the group of acquired images.

[0054] This process further includes a process of calculating a second BOT based on the calculated droplet generation rate, i.e., calculating the elapsed clock at which the droplet generation rate becomes appropriate as BOT_2 (S309).

[0055] Here, BOT_1 is controlled by driving the vibration element V to maintain a constant value, but BOT_2 varies for each second particle. One of the main causes of variation is the size of the second particles. Since the size of the second particles varies, BOT varies for each second particle. When observed at an early time point, the BOT is measured before all particles break off (the droplet generation rate is "0"). When observed at a long time point, the BOT is measured after all particles break off (the droplet generation rate is "1"). In other words, the elapsed time at which the droplet generation rate reaches an appropriate value can be considered BOT_2 and serves as a kind of indicator. Examples of this appropriate value include, but are not limited to, the elapsed time calculated backward from the droplet generation rate at which the separation results are relatively good (e.g., a droplet generation rate of 0.5±20%, preferably a droplet generation rate of 0.5±10%), but this embodiment is not limited to this.

[0056] Furthermore, the greater the variation in BOT, the longer the charging interval that encompasses all BOT, leading to a decrease in purity (the ratio of particles to be sorted among the particles to be sorted) and efficiency (the efficiency of sorting particles to be sorted). Therefore, this method is effective for particles with little variation in BOT, i.e., large-diameter particles with a uniform size of the second particles, for example, carriers that hold cells, among other particles.

[0057] Furthermore, BOT_2 can be calculated based on the calculated droplet generation rate, or it can be calculated based on the presence or absence of target particles in the droplets being observed by applying a voltage to the liquid column at each elapsed time from the reference time. In this case, the drive voltage of the vibration element V may be turned off or set to a low output beforehand, and then the drive voltage of the vibration element V may be reset. Alternatively, the above-described information may be acquired while gradually varying the drive voltage of the vibration element V, and then the drive voltage of the vibration element V may be reset.

[0058] In this step, image acquisition processing is performed only on the second particles (large diameter particles) to be sorted. This is achieved by identifying the particles to be sorted based on, for example, a threshold value for the intensity of forward scattered light. Furthermore, images, which are one type of optical information, may be excluded from the judgment target if the accumulated strobe light emission of two or more particles occurs within the exposure time of the imaging unit 151 constituting the optical information acquisition unit 15.

[0059] [Step of determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information]

[0060] In this process, parameters for separating the second particles are determined based on a comparison between the first optical information and the second optical information. Specifically, this process further includes a step of predicting a decline in separation performance based on a comparison between BOT_1 and BOT_2. More specifically, ΔBOT is calculated, and if the calculated value meets a predetermined threshold (e.g., a clock value), a decline in separation performance to the extent that the recovery rate will be 80% or less is predicted. The degree of decline in separation performance varies depending on the configuration of the device and the particle size, and therefore cannot be determined uniquely. However, once the relationship between ΔBOT and performance decline is understood, the degree of performance decline can be estimated simply by measuring ΔBOT. The same applies to ΔBOP, which will be described later.

[0061] Thereafter, if ΔBOT satisfies a predetermined threshold, it is assumed that droplet breakage due to the second particle is dominant, and setup for BOT_2 is performed. On the other hand, if the threshold is not satisfied, it is assumed that droplet breakage due to the first particle is dominant, and setup for BOT_1 is performed.

[0062] As described above, from the viewpoint of tearing stability, it is preferable that BOT_1 is constant. That is, it is preferable that this process further includes a process of controlling the parameters so that the first BOT is constant.

[0063] The parameter can be, for example, one or more of the group consisting of sheath pressure, sample pressure, piezoelectric drive output, droplet charge timing, and charge interval. Note that, in this specification, "sheath pressure" refers to the pressure applied to the sheath tank, and "sample pressure" refers to the pressure applied to the sample tube.

[0064] 5 is a diagram showing an example of a setup flow for BOT_1. First, BOT_1 is measured (S401) using the above-described method, and then the determined parameters are controlled to keep BOT_1 constant (S402). Next, the process may further include, for example, setting charge intervals before and after the first BOT (S403).

[0065] 6 is a diagram showing an example of the setup flow for BOT_2. Using the method described above, first, BOT_2 is measured (S501), and then the positional relationship between BOT_1 and BOT_2 is adjusted (S502). Next, the determined parameters are controlled to maintain BOT_1 constant (S503). Next, for example, the method may further include a step of setting a charge interval before and after the second BOT (S504). Note that in this embodiment, the charge interval may further include a step of determining the charge interval based on the rate of droplets containing the second particle.

[0066] The specific method for determining parameters (here, the charge interval) is to set the elapsed time BOT_1 relative to the reference time as the center of the charge interval. The range of the charge interval can be set within an arbitrary range from "center - (M-0.5) clocks" to "center + (N-0.5) clocks" (M and N are natural numbers).

[0067] The range of the charge interval may be determined so as to include the range in which the droplet generation rate X is 0 < X ​​< 1 in the elapsed time range searched to obtain BOT_2. This makes it possible to cover variations in BOT_2.

[0068] Alternatively, ΔBOT may be adjusted within the range of ΔBOT≧1 clock by changing the drive output of the vibration element V. After the adjustment, fine adjustment of the output of the vibration element V may be continued so that BOT_1 remains constant. This allows for the adjustment of ΔBOT to control the length of the liquid column, which may result in particles not targeted for collection being collected. The longer the length of the liquid column, the greater the likelihood that particles not targeted for collection will be present in the liquid column. Furthermore, if the break-off timing of droplets formed by driving the vibration element V occurs at the end of the charge interval, the charge amount will be incomplete, resulting in variations in flight distance. Therefore, controlling BOT_1 to a constant value can prevent droplets from scattering.

[0069] Furthermore, if it is known that particles not to be sorted are present in the droplets or liquid column that break off prior to BOT_2, a decision may be made not to sort. That is, the particle sorting method according to this embodiment may further include a step of deciding not to sort if particles not to be sorted are present in the liquid column region that breaks off during the charge interval, other than droplets that break off in the second BOT. This makes it possible to improve purity by deciding not to sort if particles not to be sorted are present in the droplets and liquid column that break off prior to BOT_2. However, efficiency decreases.

[0070] [Modification 1]

[0071] In this embodiment, the above-described adjustment may be performed in combination with a method in which droplets before and after the droplets of particles to be collected are charged multiple times (continuously charged) and collected all at once.

[0072] [Modification 2]

[0073] If ΔBOT does not meet a predetermined threshold, or the larger ΔBOT is, the piezo-controlled droplet breaks off (BOT_1 becomes dominant) before the wave generated by the progression of the second particle begins to grow, making the separation performance less likely to deteriorate. Assuming that the BOT of the droplet controlled by the vibration element V is basically adjusted to be the shortest, the wave growth associated with the progression of the particle is primarily independent of the device parameters. Therefore, in some cases, ΔBOT can be increased by adjusting the device parameters, for example, by changing (e.g., decreasing) the sheath pressure so that the break-off of BOT_1 becomes dominant and searching for droplet formation suitable for separation. This method may also improve the deterioration of separation performance.

[0074] [Modification 3]

[0075] Here, when fluorescence information of the side stream is used as the optical information, the trajectory of the side stream of the first particle and the trajectory of the side stream of the large particle (second particle) may be compared, and each BOT may be adjusted so that these follow appropriate trajectories, thereby enabling efficient sorting even when the particles to be sorted are large. Note that in this modified example, the above-mentioned effect may be achieved by using optical information of not only the side stream but also other parts.

[0076] <Adjustment using ΔBOP (break-off point)>

[0077] A method for calculating ΔBOP and predicting the deterioration of preparative separation performance will be described below.

[0078] By using this technology, as with adjustment using ΔBOT, it is possible to predict the possibility of a decline in separation performance without performing separation, thereby saving time and effort and improving separation performance.

[0079] ΔBOP=BOP_2 (hereinafter also referred to as "second BOP")-BOP_1 (hereinafter also referred to as "first BOP"), and can be determined by measuring BOP_1 and BOP_2.

[0080] [Step of supplying a first driving voltage to the vibration element V that applies vibration to the fluid]

[0081] 10 is a diagram showing an image of BOP_1. In this process, similar to the adjustment using ΔBOT, a first driving voltage is supplied to the vibration element V that vibrates the fluid, thereby vibrating the fluid and forming a first droplet suitable for collection. In other words, by controlling the vibration element V, a droplet suitable for collection and having the shortest BOT is formed.

[0082] In this embodiment, the measurement of BOP_1 does not necessarily require the above-mentioned calibration beads, because BOP_1 can be calculated without using calibration beads.

[0083] [Step of acquiring first optical information of fluid and droplets generated by vibration of the vibration element V due to the first drive voltage]

[0084] In this step, first optical information of the fluid and droplets generated by the vibrations applied in the above-described step is acquired, for example, by the optical information acquisition unit 15 described below. The optical information may be, for example, at least one of an image and fluorescent information (e.g., fluorescent information of a side stream).

[0085] This process further includes a step of determining a position where the droplet breaks off as a first BOP (break-off point) based on the first optical information. That is, BOP_1 is a position where the droplet breaks off based on the first optical information.

[0086] [Step of supplying a second driving voltage lower than the first driving voltage to the vibration element V]

[0087] Fig. 7 is a diagram showing an example of the measurement flow of BOP_2. Fig. 11 is a diagram showing an image of BOP_2. In this step, a second driving voltage lower than the first driving voltage (including the case where the driving voltage is turned off) is supplied to the vibration element V (S601).

[0088] In this embodiment, similar to the adjustment using ΔBOT, it is assumed that the second particles are “large diameter particles.” That is, BOP_2 is the position where droplets containing the second particles (large diameter particles) break off.

[0089] In this process, a sample liquid containing large-diameter particles is delivered (S602), and a second driving voltage is supplied to the vibration element V that vibrates the fluid, thereby vibrating the fluid and forming waves caused by the second particles (large-diameter particles).

[0090] [Step of acquiring second optical information of fluid and droplets containing second particles generated by vibration of the vibration element V due to the second driving voltage]

[0091] In this step, second optical information of the fluid and droplets generated by the vibrations applied in the above step is acquired, for example, by the optical information acquisition unit 15 (described later) (S603). The optical information may be, for example, at least one of an image and fluorescent information (e.g., fluorescent information of a side stream). The droplets included in the second optical information are formed by waves generated by second particles (large-diameter particles).

[0092] BOP_2 is the position where the droplet containing the second particle (large particle) breaks off. Specifically, the BOP_2 measurement method involves first observing the liquid column containing the second particle (S604). In S604, n is either X, 1, or Y, where X and Y are any natural numbers. Next, the position where the particle reaches after n droplets has passed since particle detection is photographed and observed using the imaging unit 151 (described later) (S605). When using ΔBOP, the measurement can be calculated using a length unit equivalent to one droplet cycle instead of clock units. Note that cycle length = flow velocity × clock unit time. Next, α images are acquired after n droplets have passed since particle detection (S606). Next, out-of-focus images (β images) are removed, and the droplet generation images are counted (γ images) (S607). Next, the liquid column containing the particle is observed (S608).

[0093] This process further includes a step of calculating the occurrence rate of droplets containing the second particles based on the second optical information. Specifically, as described above, optical information (here, image acquisition) of the liquid column containing the second particles is acquired. The interval of elapsed time is, for example, a length unit corresponding to one droplet cycle of driving the vibration element V. To acquire the optical information (preferably, images), the LED is strobed at the moment of the elapsed time. The imaging unit 151 is moved to a position where the arrival position of the particle can be observed at each elapsed time, and multiple images are acquired at each elapsed time, and the occurrence rate of droplets is calculated. In other words, here, the occurrence rate of droplets is calculated from the group of acquired images.

[0094] This process further includes a process of calculating a second BOP based on the calculated droplet generation rate, i.e., calculating the elapsed position where the droplet generation rate is appropriate as BOP_2 (S609).

[0095] Here, BOP_1 is controlled by driving the vibration element V so as to be a constant value, but BOP_2 varies for each second particle. One of the main causes of variation is the size of the second particles. Since the size of the second particles basically varies, the BOP varies for each second particle. When observed after an early elapsed time, the BOP is measured before all particles have broken off (the droplet generation rate is "0"). When observed after a long elapsed time, the BOP is measured after all particles have broken off (the droplet generation rate is "1"). In other words, the BOP at which the droplet generation rate is 0.4 to 0.6 (preferably around 0.5) can be said to be the average BOP_2 and serves as a kind of index, but this numerical range is not limiting in this embodiment.

[0096] In addition, the more the BOP varies, the longer the charging section that includes all the BOP becomes, leading to a decrease in purity and efficiency. Therefore, this method is effective for particles with little variation in BOP, i.e., large particles with a uniform second particle size, for example, particularly carrier particles.

[0097] In this step, image acquisition processing is performed only on the second particles (large diameter particles) to be sorted. This is achieved by identifying the target particles using a threshold value for the intensity of forward scattered light, etc. Furthermore, images, which are one type of optical information, may be excluded from the judgment target if the cumulative strobe light emission of two or more particles occurs within the exposure time of the imaging unit 151 constituting the optical information acquisition unit 15.

[0098] [Step of determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information]

[0099] In this process, parameters for separating the second particles are determined based on a comparison between the first optical information and the second optical information. Specifically, this process further includes a step of predicting a decrease in separation performance based on a comparison between BOP_1 and BOP_2. More specifically, ΔBOP is calculated, and if the value satisfies a predetermined threshold (e.g., a length corresponding to one droplet cycle), it is determined that the droplet breakage due to the second particles is predominant, and setup for BOT_2 is performed. On the other hand, if the threshold is not satisfied, it is determined that the droplet breakage due to the first particles is predominant, and setup for BOT_1 is performed.

[0100] As described above, from the viewpoint of tearing stability, it is preferable that BOP_1 be constant. That is, it is preferable that this process further includes a process of controlling the parameters so that the first BOP is constant.

[0101] The parameter can be, for example, one or more of the group consisting of sheath pressure, sample pressure, piezo drive output, droplet charge timing, and charge interval.

[0102] The setup flows for BOP_1 and BOP_2 can be obtained by replacing the setup flows for BOT_1 and BOT_2 described above with BOP_1 and BOP_2, respectively.

[0103] 2. Second embodiment (particle sorting system 1)

[0104] Figure 12 is a schematic conceptual diagram showing a first embodiment of a particle sorting system 1 according to the present technology, and Figure 13 is a schematic conceptual diagram showing a second embodiment of a particle sorting system 1 according to the present technology.

[0105] The particle sorting system 1 according to the present technology includes at least a vibration element V, a voltage supply unit 13, an optical information acquisition unit 15, and a control unit 16. In addition, as necessary, the system includes a flow path P, a light irradiation unit 11, a light detection unit 12, the vibration element V, a sorting unit 14, an analysis unit 17, a memory unit 18, a display unit 19, and the like.

[0106] Each part will be explained in detail below.

[0107] (1) Flow path P

[0108] The particle sorting system 1 according to the present technology is a system that sorts particles flowing through a flow path P. The flow path P may be provided in advance in the particle sorting system 1, but it is also possible to use a commercially available flow path P or a disposable chip provided with the flow path P.

[0109] The shape of the flow path P is not particularly limited and can be freely designed. For example, not only the flow path P formed in a substrate T made of two-dimensional or three-dimensional plastic (e.g., PP, PC, COP, PDMS, etc.) or glass as in the first embodiment shown in Fig. 12 , but also a flow path P used in a conventional flow cytometer or the like as in the second embodiment shown in Fig. 13 can be used in the particle sorting system 1 according to the present technology.

[0110] Furthermore, the width, depth, and cross-sectional shape of the flow channel P are not particularly limited as long as they are capable of forming a laminar flow, and can be freely designed. For example, a microchannel with a width of 1 mm or less can also be used in the particle sorting system 1 according to the present technology. In particular, a microchannel with a width of approximately 10 μm or more and 1 mm or less can be suitably used in the particle sorting system 1 according to the present technology.

[0111] A sample inlet P1 into which a liquid containing particles to be separated (sample liquid) is introduced, a sheath inlet P2 into which a sheath liquid is introduced, etc. are formed in the flow path P. In the first embodiment, the sample liquid is introduced from the sample liquid reservoir B1 to the sample inlet P1, and merges with the sheath liquid introduced from the sheath liquid reservoir B2 to the sheath inlet P2, and is discharged from an orifice P3 provided at the end of the flow path P.

[0112] Although not shown, a suction outlet P4 for eliminating blockages and air bubbles can also be formed in the flow path P. A negative pressure source such as a vacuum pump is connected to the suction outlet P4, and when blockages or air bubbles occur in the flow path P, the pressure inside the flow path P is made negative, causing the flow to temporarily reverse, thereby eliminating the blockages or air bubbles.

[0113] In this embodiment, the term "particle" broadly refers to biological particles such as cells, microorganisms, and ribosomes, as well as synthetic particles such as latex particles, gel particles, and industrial particles. In this technology, particles are contained in fluids such as liquid samples. Examples of biological particles include chromosomes, ribosomes, mitochondria, and organelles (subcellular organelles) that constitute various cells. Examples of cells include animal cells (e.g., blood cells), plant cells, and the like. Examples of microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Examples of biological particles also include biopolymers such as nucleic acids, proteins, and complexes thereof. Industrial particles may be, for example, organic or inorganic polymeric materials, metals, and the like. Examples of organic polymeric materials include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, and the like. Examples of inorganic polymeric materials include glass, silica, magnetic materials, and the like. Examples of metals include gold colloids, aluminum, and the like.

[0114] Furthermore, in this embodiment, the particles may be carriers (sometimes referred to as "carriers") or cells, and the carrier may hold one or more cells or a plurality of cells. For example, a biological component (e.g., a cell or a cell-derived component (e.g., a secretion)) may be held. Holding a biological component on the carrier includes, for example, a case where a biological component is captured on the carrier or a case where a biological component is encapsulated in the carrier. The carrier may be, for example, a carrier used for secretion analysis. The carrier may be an emulsion, and in this case, the particles may be recovered in a state contained in the emulsion. In this case, the recovered fractionated product may be an emulsion, and the dispersoid constituting the emulsion may be particles contained in the emulsion containing the particles to be separated. The dispersion medium constituting the emulsion may be appropriately selected by those skilled in the art, for example, depending on the type of emulsion particles. The emulsion may be a multiple emulsion. Examples of multiple emulsions include oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. The shape of the carrier is not particularly limited, and may be any particle shape such as spherical, ellipsoidal, or rod-like. Specific examples of the carrier include the above-mentioned emulsions, beads, and gel capsules.

[0115] The particles passing through the flow path P can be labeled with one or more dyes such as fluorescent dyes. In this case, examples of fluorescent dyes that can be used in this embodiment include Cascade Blue, Pacific Blue, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Propidium iodide (PI), Texas Red (TR), Peridinin chlorophyll protein (PerCP), Allophycocyanin (APC), 4',6-Diamidino-2-phenylindole (DAPI), Cy3, Cy5, Cy7, and Brilliant Violet (BV421).

[0116] (2) Light irradiation unit 11

[0117] The particle sorting system 1 according to this embodiment can be equipped with a light irradiation unit 11. The light irradiation unit 11 irradiates light onto particles flowing through the flow path P. In the particle sorting system 1 according to this embodiment, the light irradiation unit 11 is not essential, and it is also possible to irradiate light onto particles flowing through the flow path P using an external light irradiation device or the like.

[0118] The type of light irradiated from the light irradiating unit 11 is not particularly limited, but in order to reliably generate fluorescence and scattered light from the particles, light with a constant light direction, wavelength, and light intensity is preferred. Specific examples include lasers and LEDs. When a laser is used, the type is also not particularly limited, but one or more of the following lasers can be freely combined: an argon ion (Ar) laser, a helium-neon (He-Ne) laser, a dye laser, a krypton (Cr) laser, a semiconductor laser, and a solid-state laser combining a semiconductor laser with a wavelength conversion optical element.

[0119] The particle sorting system 1 according to this embodiment can also employ a so-called multi-spot to irradiate light onto multiple positions in the flow path P. In this case, although not shown, multiple light irradiating units 11 may be provided, or, although not shown, it is also possible to split light from one light irradiating unit 11 via a light control unit such as a spectroscope, and irradiate light onto multiple positions in the flow path P.

[0120] (3) Light detection unit 12

[0121] The particle sorting system 1 according to this embodiment can be equipped with a light detection unit 12. The light detection unit 12 detects optical information emitted from particles flowing through the flow path P. In the particle sorting system 1 according to this embodiment, the light detection unit 12 is not essential, and it is also possible to detect optical information emitted from particles flowing through the flow path P using an external light detection device or the like.

[0122] The light detection unit 12 that can be used in the particle sorting system 1 according to this embodiment is not particularly limited in the light detection method, as long as it can detect light signals from particles, and any light detection method used in known light detectors can be freely selected and used. Specifically, one or more light detection methods can be freely combined and used, such as those used in fluorescence measuring instruments, scattered light measuring instruments, transmitted light measuring instruments, reflected light measuring instruments, diffracted light measuring instruments, ultraviolet spectrometers, infrared spectrometers, Raman spectrometers, FRET measuring instruments, FISH measuring instruments, and various other spectrum measuring instruments, PMT arrays or photodiode arrays in which light receiving elements such as PMTs or photodiodes are arranged in a one-dimensional manner, or arrays in which multiple independent detection channels are arranged, such as two-dimensional light receiving elements such as CCDs or CMOSs.

[0123] The particle sorting system 1 according to this embodiment can also perform light detection from multiple positions in the flow path P. In this case, although not shown, multiple light detection units 12 may be provided, or although not shown, it is also possible to control the optical path of light from multiple positions in the flow path P via an optical control unit such as a mirror, so that one light detection unit 12 can perform light detection from multiple positions in the flow path P.

[0124] The location of the light detection unit 12 in the particle sorting system 1 according to this embodiment is not particularly limited as long as it can detect optical signals from particles, and can be freely designed as appropriate depending on the purpose. For example, as in the first and second embodiments shown in FIGS. 1 and 2 , it is preferable to place the light detection unit 12 on the opposite side of the flow path P from the light irradiation unit 11. By placing the light detection unit 12 on the opposite side of the flow path P from the light irradiation unit 11, the light irradiation unit 11 and the light detection unit 12 can be arranged in a more flexible configuration. Furthermore, for example, because fluorescence is emitted in directions other than the incident direction of the irradiated light, the light detection unit 12 may be placed on the same side as the light irradiation unit 11 or on a side 90 degrees to the side of the flow path P.

[0125] (4) Vibration element V

[0126] The vibration element V is as described above in "1. First embodiment (particle sorting method)" under "Step of supplying a first driving voltage to the vibration element V that applies vibration to the fluid."

[0127] The vibration element V only needs to be in contact with the flow path P, and may be provided, for example, as an internal structure of the substrate T having the flow path P, or as an internal structure of the particle sorting system 1.

[0128] (5) Voltage supply unit 13

[0129] A voltage supply unit 13 supplies a drive voltage to the vibration element V. The drive voltage of the vibration element V is supplied in accordance with a sine wave to form stable droplets, and is controlled by two parameters: a frequency (clock value) and an intensity (drive value). The specific control method is as explained above in "1. First embodiment (particle sorting method)," and therefore will not be explained here.

[0130] (6) Preparation section 14

[0131] The particle sorting system 1 according to this embodiment can include a sorting unit 14 that sorts particles. In the sorting unit 14, particles are sorted based on data analyzed by an analysis unit 17 (described later) from values ​​detected by the light detection unit 12. For example, in the sorting unit 14, particles can be sorted downstream of the flow path P based on analysis results of particle size, shape, internal structure, etc. derived from the analysis data.

[0132] Specifically, the vibration element V is used to apply vibration to the entire or part of the flow path P, thereby generating droplets from the discharge port of the flow path P. Note that by adjusting the amount of liquid sent to the flow path P, the diameter of the discharge port, the vibration frequency of the vibration element V, etc., it is possible to adjust the size of the droplets and generate droplets containing a fixed amount of microparticles.

[0133] Next, a charging unit (not shown) applies a positive or negative charge to the droplets based on the analysis results of the particle size, shape, internal structure, etc., which are obtained based on the data analyzed by the analysis unit 17. The charging unit imparts a positive or negative charge to the droplets ejected from the orifice P3, and is composed of a charging electrode and a voltage source that applies a predetermined voltage to the charging electrode.

[0134] The charging electrode can be arranged in contact with the sheath fluid and / or sample fluid flowing through the flow path P to impart an electric charge to the sheath fluid and / or sample fluid; for example, a charging electrode inlet can be provided on the substrate T that includes the flow path P, and the charging electrode can be inserted into this charging electrode inlet. The charging electrode may be arranged to contact the sample fluid, the sheath fluid, or both the sample fluid and the sheath fluid. However, considering the effect on particles to be sorted (especially cells, etc.), it is preferable to arrange the charging electrode to contact the sheath fluid.

[0135] In this way, by charging desired droplets with positive or negative charges, it becomes possible to separate droplets containing desired particles by electrical force. Furthermore, by synchronizing the timing of charging by the charging unit with the voltage supplied to the vibration element V, it becomes possible to charge only desired droplets.

[0136] The charged droplets are then sorted and deflected to a desired direction by deflection plates 141. Specifically, for example, deflection plates 141a and 141b deflect the direction of each droplet in the fluid stream S by an electrical force acting between the droplets and the charge applied thereto, and guide the droplets to a predetermined collection container, and are arranged opposite each other across the fluid stream S. For example, commonly used electrodes can be used for these deflection plates 141a and 141b.

[0137] A different positive or negative voltage is applied to each of the deflection plates 141 a and 141 b, and when charged droplets pass through the electric field formed by this, an electric force (Coulomb force) is generated, and each droplet is attracted toward one of the deflection plates 141 a and 141 b. In the particle sorting system 1, by changing the positive or negative charge on the droplets and the amount of charge, it is possible to control the direction of the flow (side stream) of droplets attracted by the electric field, making it possible to simultaneously sort multiple different particles.

[0138] The droplets whose paths have been changed to the desired directions by the deflection plates 141a and 141b are collected in collection containers 142a to 142c. General-purpose plastic tubes, glass tubes, or the like for experimental use can be used as collection containers 142a to 142c. These collection containers 142a to 142c are preferably replaceable within the system. Furthermore, a drainage path for the collected droplets may be connected to one of collection containers 142a to 142c that receives particles not to be sorted.

[0139] There is no particular limitation on the number of collection containers arranged in the particle sorting system 1. For example, when more than three collection containers are arranged, each droplet may be guided to and collected in one of the collection containers depending on the presence or absence and magnitude of the electrical force between the deflection plates 141 a and 141 b.

[0140] (7) Optical information acquisition section 15

[0141] The optical information acquiring unit 15 acquires optical information of the fluid and droplets D ejected from the orifice P3 of the flow path P. The optical information may be, for example, at least one of an image and fluorescent information (e.g., fluorescent information of a side stream).

[0142] The optical information acquisition unit 15 is composed of an imaging unit 151 that captures an image of the fluid or droplets D, a position adjustment mechanism 152 for adjusting the position of the imaging unit 151 to follow variations in the BOP (break-off point), and a data processing unit 153 that acquires optical information of the fluid or droplets D from the captured image. The imaging unit 151 may be an imaging device such as a CCD or CMOS camera, or various imaging elements such as a photoelectric conversion element. The particle sorting system 1 according to this embodiment may be provided with a light source (not shown) that illuminates the imaging area in addition to the imaging unit 151.

[0143] The data processing unit 153 may be configured with an information processing device including, for example, a general-purpose processor, a main memory device, an auxiliary memory device, etc. In this case, optical information can be obtained by inputting data representing image data showing the state of the fluid or droplet D captured by the imaging unit 151 into the data processing unit 153 and executing a programmed control algorithm. Such a computer program may be stored in a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, and may also be distributed via a wired or wireless network.

[0144] (8) Control unit 16

[0145] The control unit 16 determines parameters for sorting the second particles based on a comparison between the first optical information and the second optical information. The control unit 16 is configured, for example, with an information processing device including a general-purpose processor, a main memory device, an auxiliary memory device, etc. The specific method for determining the parameters is as explained above in "1. First embodiment (particle sorting method)," and therefore will not be explained here.

[0146] The control unit 16 receives the optical information obtained by the optical information acquisition unit 15 and executes a programmed control algorithm, thereby automatically controlling the parameters. Such a computer program may be stored on a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, or may be distributed via a network.

[0147] (9) Analysis section 17

[0148] The particle sorting system 1 according to this embodiment may further include an analysis unit 17, as necessary. The analysis unit 17 is connected to the light detection unit 12 and analyzes optical information detected from the microparticles by the light detection unit 12.

[0149] The analysis unit 17 calculates the feature quantities of each particle from, for example, the optical information of the light received from the light detection unit 12. Specifically, the analysis unit 17 calculates the feature quantities indicating the size, shape, internal structure, etc. of the particle from the detected values ​​of the received fluorescence and scattered light.

[0150] The analysis unit 17 is not essential to the particle sorting system 1 according to this embodiment, and it is also possible to analyze the state of particles using an external analysis device or the like based on the optical information detected by the light detection unit 12. For example, the analysis unit 17 may be implemented by a personal computer or a CPU, or may be stored as a program in hardware resources including a recording medium (e.g., non-volatile memory (USB memory), HDD, CD, etc.) and operated by the personal computer or the CPU. The analysis unit 17 may also be connected to each part of the particle sorting system 1 via a network.

[0151] (10) Storage unit 18

[0152] The particle sorting system 1 according to this embodiment can be provided with a memory unit 18 for storing various types of information. The memory unit 18 can store all kinds of information, such as information data detected by each light detection unit 12, optical information data acquired by the optical information acquisition unit 15, control data in the control unit 16, analysis data generated by the analysis unit 17, and data on particles sorted by the sorting unit 14.

[0153] In the particle sorting system 1 according to this embodiment, the storage unit 18 is not essential, and an external storage device may be connected. As the storage unit 18, for example, a hard disk or the like can be used.

[0154] (11) Display section 19

[0155] The particle sorting system 1 according to this embodiment can be provided with a display unit 19 that displays various information. The display unit 19 can display all sorts of information, such as information data detected by each light detection unit 12, optical information data acquired by the optical information acquisition unit 15, control data in the control unit 16, analysis data generated by the analysis unit 17, and data on particles sorted by the sorting unit 14.

[0156] In the particle sorting system 1 according to this embodiment, the display unit 19 is not essential, and an external display device may be connected. As the display unit 19, for example, a display or a printer may be used.

[0157] (12) Other

[0158] In addition, the particle sorting system 1 according to this embodiment may have the flow path P, light irradiation unit 11, light detection unit 12, vibration element V, optical information acquisition unit 15, analysis unit 17, memory unit 18, display unit 19, etc., arranged independently as necessary. For example, the flow path P may be provided in advance in the particle sorting system 1, but it is also possible to install a commercially available flow path P or a disposable chip provided with the flow path P in the particle sorting system 1 and perform analysis or sorting.

[0159] The light irradiation unit 11 and the light detection unit 12 may be provided in advance in the particle sorting system 1, but it is also possible to use an external light irradiation device or external light detection device to irradiate light onto or detect light from particles flowing through the flow path P. Furthermore, the optical information acquisition unit 15, the analysis unit 17, the memory unit 18, the display unit 19, etc. may be provided in advance in the particle sorting system 1, but it is also possible to use external detection devices, analysis devices, storage devices, display devices, etc. In this case, each device may be connected via a wired or wireless network.

[0160] 3. Third embodiment (program)

[0161] The program according to this embodiment is a program that causes the particle sorting system 1 to perform functions equivalent to those performed by the voltage supply unit 13, optical information acquisition unit 15, and control unit 16 in "2. Second embodiment (particle sorting system 1)" described above. Note that this program can also be distributed independently.

[0162] The program according to the present embodiment may be provided in a state stored on a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, and may be downloaded to a computer or the like for use. Alternatively, the program according to the present embodiment may be distributed from outside via a network such as the Internet and downloaded to a computer or the like for use.

[0163] The electronic computer to which the program according to this embodiment has been downloaded acquires information about ΔBOT or ΔBOP, executes the control algorithm of the downloaded program, and determines the parameters. The electronic computer issues commands to particle sorting system 1 based on the determined parameters, thereby automatically controlling the parameters of particle sorting system 1.

[0164] The present technology may also employ the following configurations. [1] A particle sorting method comprising the steps of: supplying a first drive voltage to a vibration element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element at the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element at the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information. [2] The particle sorting method according to [1], wherein the optical information is at least one of an image and fluorescent information. [3] The particle sorting method according to [1] or [2], wherein the droplets included in the second optical information are formed by waves generated by the second particles. [4] The particle sorting method according to [1], wherein the first optical information relates to first particles different in size from the second particles. [5] The particle sorting method according to [4], wherein the second particles have a larger particle size than the first particles. [6] The particle sorting method according to [4] or [5], further comprising a step of determining a time until a droplet containing the first particle breaks off relative to a reference time based on the first optical information as a first BOT (break-off time). [7] The particle sorting method according to [6], further comprising a step of calculating a generation rate of droplets containing the second particle based on the second optical information. [8] The particle sorting method according to [7], further comprising a step of observing droplets that have been deflected or not deflected by charging by applying a voltage to a liquid column for each elapsed time from the reference time, and calculating a second BOT based on the calculated generation rate of droplets or the presence or absence of particles to be sorted in the droplets being observed. [9] The particle sorting method according to [8], further comprising a step of predicting a deterioration in sorting performance based on a comparison between the first BOT and the second BOT.

[10] The particle sorting method according to [9], further comprising the step of controlling the parameters so that the first BOT is constant.

[11] The particle sorting method according to

[10] , wherein the parameter is any one or more of the group consisting of sheath pressure, sample pressure, piezoelectric drive output, droplet charge timing, and charge interval.

[12] The particle sorting method according to

[11] , further comprising a step of setting the charge interval before and after the first BOT.

[13] The particle sorting method according to any one of [1] to

[12] , further comprising a step of determining the charge interval based on a generation rate of droplets containing the second particle.

[14] The particle sorting method according to

[11] , further comprising a step of determining not to perform sorting when particles other than droplets that break off at the second BOT are present in a liquid column region that breaks off during the charge interval and are not to be sorted.

[15] The particle sorting method according to [1], further comprising a step of setting a position up to when the droplet breaks off as a first BOP (break-off point) based on the first optical information.

[16] The particle sorting method according to

[15] , further comprising a step of calculating a generation rate of droplets containing the second particle based on the second optical information.

[17] The particle sorting method according to

[16] , further comprising a step of calculating a second BOP based on the calculated generation rate of droplets.

[18] The particle sorting method according to

[17] , further comprising a step of predicting a degradation in sorting performance based on a comparison between the first BOP and the second BOP.

[19] A particle sorting system comprising: a voltage supply unit that supplies a first drive voltage to a vibration element that imparts vibration to a fluid; and an optical information acquisition unit that acquires first optical information of the fluid and droplets generated by the vibration of the vibration element with the first drive voltage, wherein the voltage supply unit supplies a second drive voltage lower than the first drive voltage to the vibration element; the optical information acquisition unit acquires second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element with the second drive voltage; and a control unit that determines parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[20] A program that executes the following steps: supplying a first drive voltage to a vibration element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element at the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element at the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0165] The present technology will be described in more detail below based on examples. Note that the examples described below are examples of typical examples of the present technology, and the scope of the present technology should not be construed as being narrow.

[0166] Example 1: Automatic adjustment using ΔBOT

[0167] [Calculating ΔBOP and Automated Adjustment to Predict Deterioration in Preparative Performance]

[0168] (Summary) In this example, the value of ΔBOT can be used to predict, to some extent, whether or not sorting performance will decline. In droplet-based cell sorters, droplets are typically controlled to minimize BOT_1. However, if the clock value of the vibrating element V at ΔBOT is below a predetermined threshold, interference between two waves may occur, potentially making droplet breakoff unstable. Simply applying a charge during the time interval before and after BOT_1 may result in a decrease in yield. Alternatively, if BOT_2 is shorter than BOT_1, applying a charge based on BOT_1 as usual will not be enough to apply a charge to droplets containing second particles (larger particles), resulting in no sorting (because they have already broken off at BOT_2).

[0169] The threshold value is set appropriately depending on the cell sorter, the orifice diameter used, and the particle size. The threshold value is based on the interval from BOT_2 back to the point at which waves generated by the movement of particles begin to be observed.

[0170] (Adjustment Example) In this example, a particle sorting system 1 is used that is configured to detect scattered light or fluorescence generated by laser irradiation of flowing particles. The timing of particle detection is defined as the "reference time." The particle sorting system 1 has an optical information acquisition unit 15 that observes the liquid column, which is movable in the direction of the liquid column's movement. An LD or LED is provided on the opposite side, and it has a control function that allows it to emit light at any timing and for any emission time.

[0171] Next, the vibration element V is controlled to form a droplet suitable for sorting and with the shortest BOP. The time from the reference time until the droplet containing the first particle breaks off is defined as BOT_1 (see FIG. 8). In this example, calibration beads are used to measure BOT_1.

[0172] Next, the output of the vibration element V is turned OFF or at low output, and the output of the vibration element V is adjusted by low output drive to the extent that droplets are formed by the vibration element V. Note that when the output of the vibration element V is turned OFF, droplet formation by natural jetting becomes dominant. In this case, since there is a possibility that the scattering of droplets may contaminate the inside of the device, it is preferable to drive the vibration element V at a minimum level so that droplet formation by driving the vibration element V becomes dominant. However, if there is no need to consider the possibility that the scattering of droplets may contaminate the inside of the device, the output of the vibration element V may be turned OFF.

[0173] The output of the vibration element V supplies a drive voltage that is, for example, 1% to 20%, preferably 5% to 15%, of the drive voltage during normal droplet control drive. The time from the reference time until the droplet containing the second particle (large particle) breaks off is defined as BOT_2 (see FIG. 9).

[0174] A specific method for measuring BOT_2 involves moving the imaging unit 151 to the liquid column containing the second particle in accordance with the elapsed time from the reference time and performing observation (see FIG. 14 ). In this case, if the clock phase at the time of detection of the second particle is Φx, the range of Φx may be limited, and observation of the liquid column may be performed only for second particles detected within a specified phase range (e.g., a range of the central 20% phase of one wavelength, preferably a range of the central 10% phase of one wavelength). In liquid column observation, the position of the second particle varies by one wavelength. Therefore, limiting the phase suppresses variation in the observed particle position, making it easier to capture the particle in the center of the observed image.

[0175] The elapsed time interval is, for example, one clock cycle of the vibration element V. To acquire an image, the LED positioned opposite the optical information acquisition unit 15 is strobed at the moment the elapsed time has elapsed. The imaging unit 151 must predict the position where the second particle will arrive at each elapsed time and move it appropriately so that the waves and droplets caused by the particle are captured in the image. Multiple liquid column images are acquired at each elapsed time, and the droplet generation rate is calculated (see Figure 15). The experimental conditions used were a cell sorter (SH800, manufactured by Sony Corporation), a 130 μm M1 chip (manufactured by Sony Corporation), 40 μm Beads as the sample, and a setting mode of Single 3 drops. An approximate straight line is drawn on the plot of elapsed time vs. droplet generation rate, and the elapsed time of the droplet generation rate is then calculated based on this to obtain BOT_2. The occurrence of droplets can be determined, for example, by determining the number of liquid masses (black areas in the image) with a certain area or larger in the image through image processing (see Figure 16).

[0176] Next, second particles (large diameter particles) to be sorted are gated based on FSC (forward scattered light), BSC (backward scattered light), SSC (side scattered light), fluorescence, etc. Optical information (here, an image) of the liquid column containing the gated particles is acquired.

[0177] When acquiring an image, if the strobe light of two or more particles is accumulated within the exposure time of the image capturing unit 151, the image will be blurred (see FIG. 17), and therefore will be excluded from the calculation of the droplet occurrence rate. Such images occur probabilistically according to the frame rate of the image capturing and the particle EPS (events per second) (theoretically, the particle arrival interval is considered to be an event that follows a Poisson process).

[0178] Images that capture one particle and images that capture multiple particles can be distinguished, for example, based on the average brightness value of each image (see FIG. 18). The average brightness value is calculated for each acquired image, and images near the Min value are images that capture one strobe (one particle). Therefore, images in the range from the Min value to the coefficient * Min value are selected and can be used as references for calculating the droplet occurrence rate. Bright images outside this range are deemed to have captured multiple strobes (multiple particles) and are therefore excluded from the determination of the droplet occurrence rate.

[0179] Next, ΔBOT is calculated as ΔBOT = BOT_2 - BOT_1. For example, in this example, if ΔBOT is 7.2 clocks or less, a decrease in sorting performance is predicted (see Figure 19). Figure 19 is a plot of the recovery rate (Recovery) of 30 μm beads vs. ΔBOT. It can be seen that the recovery rate begins to decrease when ΔBOT falls below 0.6 ms. Since the piezo clock at this time was 12 kHz, 0.6 ms is equivalent to 7.2 clocks. Note that in this example, 7.2 clocks was set as the predetermined threshold value of the vibration element V at ΔBOT, but this is merely an example.

[0180] Automatic adjustment for droplet sorting triggered by particle progression

[0181] (Summary) In this embodiment, droplets induced by particle movement can be targeted for collection. Here, if interference between two types of waves is a factor in reduced sorting performance, it is preferable to create a state in which one of the waves is dominant. Assuming that BOT_1 is already controlled to a point close to the shortest distance, and BOT_2 is not a phenomenon primarily dependent on device control, there is no obvious way to make BOT_1 dominant in the interference state of the two types of waves. Therefore, to create a state in which BOT_2 is dominant, the drive output of the vibration element V is intentionally reduced and BOT_1 is lengthened. The shorter BOT_2 is relative to BOT_1, the more dominant BOT_2 becomes in the break-off timing. In a state in which BOT_2 is dominant, droplets induced by particle movement can be targeted for collection by applying charge in the time interval before and after BOT_2.

[0182] (Adjustment Example) Execute the process of "[Automatic adjustment to calculate ΔBOT and predict degradation of preparative separation performance]." Next, for each particle, the range of the charge period can be set within an arbitrary range from "center - (M - 0.5) clock" to "center + (N - 0.5) clock" based on the timing when the elapsed time from the reference time becomes BOT_2 (M, N: natural numbers).

[0183] The specific length of the charge section may be determined so as to encompass the range in which the droplet generation rate X was 0 < X ​​< 1 within the elapsed time range searched to determine BOT_2. The greater the variation in the size of the particles to be separated, the wider this range becomes, leading to a decrease in efficiency. Furthermore, the greater the variation in the break-off position, the greater the possibility of variation in the droplet flight distance during separation, so a smaller variation is preferable. Therefore, this method of separating droplets generated as the particles move is particularly effective for carrier particles with a uniform particle size.

[0184] Furthermore, the drive output of the vibration element V may be changed to adjust ΔBOT within the range of "ΔBOT≧1 clock." After adjustment, the piezo output may be finely adjusted so that BOT_1 remains constant (by appropriately controlling BOT_1, it is possible to prevent droplets that break off at the BOP_1 position from scattering over a wide area). Note that the charging of the liquid column requires a certain amount of time for the rise and fall, and droplets that break off during this period will have an incomplete amount of charge, which can lead to variations in the landing positions of the droplets if not appropriately controlled.

[0185] A droplet that breaks off before BOT_2 during charging into the liquid column may be collected together with droplets that break off at BOT_2. Therefore, if there are particles other than droplets that break off at BOT_2 in the liquid column region that breaks off during charging that are not to be collected, the control unit 16 may decide not to collect the particles (see FIG. 20).

[0186] Example 2: Automatic adjustment using ΔBOP

[0187] (Summary) In the first embodiment described above, automatic adjustment is performed using ΔBOT, but in the present technology, ΔBOP may be used instead of ΔBOT.

[0188] (Adjustment example) Calculate as follows: ΔBOP = BOP_2 - BOP_1. Note that while ΔBOT was determined in clock units to predict the decline in separation performance, ΔBOP can be calculated by converting it into a length unit equivalent to one droplet cycle.

[0189] <Example 3: Setting of charging section using multi-drop>

[0190] (Overview) FIG. 21 is a diagram illustrating the concept of multi-drop. In this specification, "multi-drop" refers to a function that allows the number of charged droplets before and after the Best Delay determined by calibration to be changed. In this Example 3, by performing adjustments using multi-drop, even if the Best Delay timing of the first particle remains the same, the charge interval can be extended to include the BOP of the second particle, thereby covering variations in the tearing position (or uniforming the total charge amount of droplets that coalesce during flight). This can increase the recovery rate of the second particle (large particle). In other words, even if the second particle is not observed, if the recovery rate when the second particle is collected under the conditions determined for the first particle is poor, or if afterimages (jitter) are observed when observing the droplet state after the second particle is delivered, the recovery rate of the second particle can be improved by increasing the charge interval (number of charged droplets). However, since increasing the number of charged droplets has the disadvantage of increasing the number of aborts, the charge interval must be set appropriately.

[0191] (Adjustment Example) Figures 22 to 24, 26, and 28 are diagrams illustrating an actual case where multi-drop is performed. In Figures 22 to 24, 26, and 28, "column" refers to the "liquid column portion," "particle" refers to the "second particle," and "drop" refers to the "liquid droplet." The horizontal axis indicates the number of clock units, and the vertical axis indicates the number of droplet cycles. Furthermore, the area within the bold frame in each figure indicates the area to be monitored. Furthermore, in Figures 22 to 24, 26, and 28, charged second particles and droplets are represented by circles.

[0192] Fig. 22 shows the case where the particles are collected in the single-droplet charging mode, and Fig. 23 shows the case where the particles are collected in the three-droplet charging mode. Fig. 22 and Fig. 23 show examples of normal particles that do not experience critical shredding, and the timing of charging should be around the time when the target particles reach the BOP.

[0193] Figure 24 shows a three-drop charging mode with low drive, critical breakage (Pa) of 15, PZT (piezoelectric element) breakage (Pi) of 18, and ΔBOP (N) = 3. Using the same concept as above, if the three-drop charging mode is used with critical breakage as the charging standard, as shown in Figure 25, there is a possibility that droplets will be charged outside the monitoring area (outside the bold frame) (i.e., a contamination risk will occur). For low drive fractionation that prioritizes efficiency, a three-drop charge is used based on BOT_2. Specifically, the charge interval can be set using the method described in the flowchart in Figure 6 above.

[0194] Figure 26 shows a three-drop charging mode with low drive, critical breakage (Pa) of 15, PZT (piezoelectric element) breakage (Pi) of 18, and ΔBOP (N) = 3. In other words, to avoid the situation shown in Figure 25, the PZT breakage (Pi) remains at 18, and the charge interval is set to cover the critical breakage (Pa) with multi-drop. By observing the BOP (Pa) of the second particle and covering the critical breakage (Pa) with multi-drop, ideal charging can be achieved. In addition, in low drive fractionation where purity is emphasized, multi-charge is used to include BOT_2, with BOT_1 (calculated at S101 in Figure 2) as the reference. Specifically, as shown in the flowchart of Figure 27, steps S701 to S703 use a method that conforms to steps S501 to S503 of the flowchart described in Figure 6 above, and in step S704, the preceding charge interval is set to include BOT_2 based on BOT_1 obtained in step S101 of Figure 2.

[0195] Figure 28 illustrates a case where the particle velocity is particularly slow. In this case, subsequent droplets will catch up with and collide with the first droplet during flight, so it is effective to charge the subsequent droplets in anticipation of this. Therefore, in the case shown in Figure 28, the 8-droplet charging mode is used. Note that, particularly for the second particle (large diameter particle) with a relatively slow particle velocity, after BOT_2 is calculated, low drive setting is not used. Instead, multi-charging is used to include BOT_2 based on BOT_1 (calculated at S101 in Figure 2) and subsequent sections. Specifically, as shown in the flowchart in Figure 29, S801 uses the method in accordance with S501 in the flowchart described above in Figure 6, and in S802, the preceding charge section is set to include BOT_2 based on BOT_1 calculated at S101 in Figure 2, and the subsequent charge section is also set to include multiple droplets.

[0196] (Experimental Example) Figure 30 is a diagram showing the results when second particles were collected using a multi-droplet. The vertical axis shows Recovery (recovery rate), and the horizontal axis shows Current (when collected in a three-droplet charging mode) and New (when sorted using a wide multi-droplet before and after; the collection method shown in Figure 28 above was implemented). The second particles collected in this experimental example were large particles with diameters of 50 μm to 70 μm. The results of this experimental example showed that the recovery rate of large particles increased by setting a charging section using a multi-droplet.

[0197] 1 Particle sorting system P Flow path P1 Sample inlet P2 Sheath inlet P3 Orifice 11 Light irradiation unit 12 Light detection unit V Vibration element 13 Voltage supply unit 14 Sorting unit 141a, 141b Deflection plates 142a to 142c Collection container 15 Optical information acquisition unit 151 Imaging unit 152 Position adjustment mechanism 153 Image data processing unit BOT Break-off time BOP Break-off point 16 Control unit D Droplet 17 Analysis unit 18 Memory unit 19 Display unit

Claims

1. A particle sorting method comprising the steps of: supplying a first drive voltage to a vibration element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element at the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element at the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

2. The particle sorting method according to claim 1, wherein the optical information is at least one of an image and fluorescent information.

3. The particle sorting method according to claim 1, wherein the droplets contained in the second optical information are formed by waves generated by the second particles.

4. The particle sorting method according to claim 1, wherein the first optical information relates to a first particle having a size different from that of the second particle.

5. The particle sorting method according to claim 4, wherein the second particles have a larger particle size than the first particles.

6. The particle sorting method described in claim 4, further comprising a step of determining the time until a droplet containing the first particle breaks off relative to a reference time based on the first optical information as a first BOT (break-off time).

7. The particle sorting method according to claim 6, further comprising the step of calculating the rate of occurrence of droplets containing the second particles based on the second optical information.

8. The particle sorting method described in claim 7, further comprising a step of observing droplets that have been deflected or not deflected by charging by applying a voltage to the liquid column at each elapsed time from the reference time, and calculating a second BOT based on the presence or absence of particles to be sorted in the droplets being observed.

9. The particle sorting method according to claim 8, further comprising the step of predicting a decrease in sorting performance based on a comparison between the first BOT and the second BOT.

10. The particle sorting method according to claim 9, further comprising the step of controlling the parameters so that the first BOT is constant.

11. The particle sorting method according to claim 10, wherein the parameter is at least one of the group consisting of sheath pressure, sample pressure, piezo drive output, droplet charge timing, and charge interval.

12. The particle sorting method according to claim 11, further comprising the step of setting the charging section before and after the first BOT.

13. The particle sorting method according to claim 1, further comprising a step of determining the charging section based on the rate of occurrence of droplets containing the second particles.

14. A particle sorting method as described in claim 11, further comprising a step of determining not to sort if particles other than droplets that break off in the second BOT and that are not to be sorted are present in the liquid column region that breaks off during the charging section.

15. The particle sorting method according to claim 1, further comprising a step of determining the position up to when the droplet breaks off as a first BOP (break-off point) based on the first optical information.

16. The particle sorting method according to claim 15, further comprising a step of calculating the rate of occurrence of droplets containing the second particles based on the second optical information.

17. The particle sorting method according to claim 16, further comprising the step of calculating a second BOP based on the calculated droplet generation rate.

18. The particle sorting method according to claim 17, further comprising the step of predicting a decrease in sorting performance based on a comparison between the first BOP and the second BOP.

19. A particle sorting system comprising: a voltage supply unit that supplies a first drive voltage to a vibration element that imparts vibration to a fluid; and an optical information acquisition unit that acquires first optical information of the fluid and droplets generated by the vibration of the vibration element with the first drive voltage, wherein the voltage supply unit supplies a second drive voltage lower than the first drive voltage to the vibration element; the optical information acquisition unit acquires second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element with the second drive voltage; and a control unit that determines parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

20. A program that executes the steps of: supplying a first drive voltage to a vibration element that imparts vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibration element at the first drive voltage; supplying a second drive voltage lower than the first drive voltage to the vibration element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibration element at the second drive voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

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