Particle sorting method, particle sorting system, and program

Abstract

This invention provides a technique for effectively sorting particles even when the particles to be sorted are relatively large. For example, a particle sorting method is provided, comprising: supplying a first driving voltage to a vibrating element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage; supplying a second driving voltage lower than the first driving voltage to the vibrating element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibrating element under the second driving voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

Description

Technical Field

[0001] This technology relates to particle sorting methods, particle sorting systems, and programs. Background Technology

[0002] In related technologies, flow cytometry has been used as a method for analyzing (in this technology, analysis includes the concept of resolution) or sorting particles such as cells, microorganisms, and ribosomes. The apparatus used for this flow cytometry is called a flow cytometer (FCM). Within a flow cytometer, the device used for sorting particles is specifically called a cell sorter.

[0003] In flow cytometry, particles flowing through a flow path are illuminated with laser light of a specific wavelength. A detection unit converts and digitizes the fluorescence, forward scattered light, and side scattered light emitted by each particle into electrical signals. Statistical analysis of the results determines the type, size, and structure of each particle. Target particles can be sorted by applying charge using vibrating elements, controlling their trajectory using deflection plates, and guiding them to a target recovery container.

[0004] In the sorting of large-diameter particles, it is known that the yield of large-diameter particles decreases. This is due to factors such as droplet turbulence and charge resistance caused by particles located between droplets. In contrast, Patent Documents 1 and 2 disclose a technique for extending the range of charge applied to particles (the so-called charged range) when the target particles to be sorted are large-diameter particles.

[0005] [List of Citations]

[0006] [Patent Literature]

[0007] Patent Document 1: JP 2019-063798 A

[0008] Patent Document 2: JP 2015-152439 A Summary of the Invention

[0009] [Technical Issues]

[0010] However, in the sorting technology for large-diameter particles, methods to improve the situation of droplet shape disorder and decreased sorting performance during particle delivery are generally unclear.

[0011] Therefore, the main objective of this technology is to provide a method that can efficiently sort even large target particles.

[0012] [Solution to the problem]

[0013] This technology first provides a particle sorting method, including: supplying a first driving voltage to a vibrating element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage; supplying a second driving voltage lower than the first driving voltage to the vibrating element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibrating element under the second driving voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0014] This technology also provides a particle sorting system, comprising: a voltage supply unit configured to supply a first driving voltage to a vibrating element configured to apply vibration to a fluid; and an imaging unit configured to acquire first optical information of fluid and droplets generated by the vibration of the vibrating element under the first driving voltage, wherein the voltage supply unit is configured to supply a second driving voltage lower than the first driving voltage to the vibrating element, and the imaging unit is configured to acquire second optical information of fluid and droplets containing second particles generated by the vibration of the vibrating element under the second driving voltage, and the particle sorting system further comprises a control unit, the control unit determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0015] This technology further provides a procedure for performing the following operations: supplying a first driving voltage to a vibrating element that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage; supplying a second driving voltage lower than the first driving voltage to the vibrating element; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibrating element under the second driving voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0016] In this specification, generally, in a cell sorter, "large diameter particles" are particles with a diameter exceeding about 1 / 5 of the orifice diameter, which is a particle size that can be stably sorted, and specific examples include particles with a diameter of about 10 μm to about 100 μm. Attached Figure Description

[0017] Figure 1 It is a schematic diagram illustrating the relationship between the combined wave formed by the wave generated by the vibrating element V and the wave generated by the large-diameter particle, and the droplet generation position.

[0018] Figure 2This is a diagram illustrating an example of the overall process of the particle sorting method according to this technique.

[0019] Figure 3 This is a diagram illustrating an example of the measurement process for BOT_1.

[0020] Figure 4 This is a diagram illustrating an example of the measurement process for BOT_2.

[0021] Figure 5 This is a diagram illustrating an example of the setup process for BOT_1.

[0022] Figure 6 This is a diagram illustrating an example of the setup process for BOT_2.

[0023] Figure 7 This is a diagram illustrating an example of the measurement process for BOP_2.

[0024] Figure 8 This is a diagram illustrating the image of BOT_1.

[0025] Figure 9 This is a diagram illustrating the image of BOT_2.

[0026] Figure 10 This is a diagram illustrating the image of BOP_1.

[0027] Figure 11 This is a diagram illustrating the image of BOP_2.

[0028] Figure 12 This is a schematic conceptual diagram illustrating a first embodiment of a particle sorting system 1 according to the present technology.

[0029] Figure 13 This is a schematic conceptual diagram illustrating a second embodiment of the particle sorting system 1 according to the present technology.

[0030] Figure 14 This is a diagram illustrating an example of imaging a portion of a liquid column containing particles at each elapsed time (clock unit).

[0031] Figure 15 This is a graph showing the ratio of droplet formation (M / N) to recovery over time (delay).

[0032] Figure 16 This is an example diagram illustrating the method for determining the state before and after droplet formation.

[0033] Figure 17 This is an example of an image representing one particle (one strobe) and an example of an image representing multiple particles (multiple strobes).

[0034] Figure 18 This is an example illustration representing image selection based on the average brightness value of each image.

[0035] Figure 19 This is a graph showing the recovery rate of 30μm beads against ΔBOT.

[0036] Figure 20 It is a diagram showing an image of charged regions that may be sorted together with droplets originating from particles.

[0037] Figure 21 This is a diagram illustrating the concept of multiple drops.

[0038] Figure 22 This is a diagram illustrating Example 3.

[0039] Figure 23 This is a diagram illustrating Example 3.

[0040] Figure 24 This is a diagram illustrating Example 3.

[0041] Figure 25 This is a diagram showing the state of a charged droplet generated outside the monitoring area (outside the thick frame).

[0042] Figure 26 This is a diagram illustrating Example 3.

[0043] Figure 27 This means that in Figure 26 The flowchart shows the method for setting the charged region in the case shown.

[0044] Figure 28 This is a diagram illustrating Example 3.

[0045] Figure 29 This means that in Figure 28 The flowchart shows the method for setting the charged region in the case shown.

[0046] Figure 30 This is a diagram showing the results when the second particle is sorted using multiple drops. Detailed Implementation

[0047] The following description, with reference to the accompanying drawings, outlines suitable methods for implementing the present technology. The embodiments described below are examples of representative embodiments of the present technology, and the scope of the present technology should not be narrowly interpreted in light of these embodiments. Note that the description will proceed in the following order.

[0048] 1. First embodiment (particle sorting method) (1) Background Technology

[0050] (2) Overview of this technology

[0051] (3) Details of the particle sorting method involved in this embodiment

[0052] Adjustment using ΔBOT (break time)

[0053] Adjustment using ΔBOP (breakpoint)

[0054] 2. Second embodiment (particle sorting system 1)

[0055] (1) Flow path P

[0056] (2) Light irradiation unit 11

[0057] (3) Optical detection unit 12

[0058] (4) Vibrating element V

[0059] (5) Voltage supply unit 13

[0060] (6) Sorting Unit 14

[0061] (7) Optical information acquisition unit 15

[0062] (8) Control Unit 16

[0063] (9) Analysis Unit 17

[0064] (10) Storage Unit 18

[0065] (11) Display unit 19

[0066] (12) Other

[0067] 3. Third Implementation Example (Program)

[0068] 1. First embodiment (particle sorting method) (1) Background Technology

[0070] As mentioned above, cell sorters are typically capable of handling particles up to about 1 / 5 of the orifice diameter; sorting particles larger than this diameter may lead to a decrease in yield. Possible reasons include droplet shape disorder and charged resistance caused by particles located between droplets.

[0071] However, methods to improve the situation where droplet shape disorder during particle delivery leads to a decline in sorting performance are generally unclear. The degree of decline in sorting performance varies depending on the device and various conditions and states of the particles, and even if parameter adjustments are made to improve recovery, the sorting performance cannot be determined without actually performing sorting to confirm whether the effect is present or not.

[0072] One method to improve sorting performance is to increase the recovery rate of particles located at the center of the droplet. However, although particles at the center of the droplet can be selected and sorted, it is impossible to ensure that all particles are present at the center of the droplet, and particles located at the edge of the droplet are discarded. Therefore, although the recovery rate is improved, the overall yield inevitably decreases.

[0073] (2) Overview of this technology

[0074] This technology is based on the following discovery: by observing the liquid column containing large-diameter particles (the arrival position of the particles) during the delivery of large-diameter particles, in relation to the elapsed time from particle detection from the laser, it was found that waves and droplets are generated in the liquid column as the particles travel, as a phenomenon independent of the driving voltage control.

[0075] Figure 1 This is a schematic diagram illustrating the relationship between the combined wave formed by the wave generated by the vibrating element V and the wave generated by the large-diameter particle, and the droplet generation location. Figure 1 The left figure illustrates the droplet generation position when the vibrating element is driven on and off. Figure 1 The middle figure illustrates the state of droplet generation position when the driving force of the vibrating element is off and the particle size changes. Figure 1 The right-hand figure schematically illustrates the droplet generation position under the combined wave state when the vibrating element is driven on and the particle size changes. In other words, Figure 1 The left figure illustrates the droplet generation position obtained by controlling the driving voltage. Figure 1 The middle figure illustrates the droplet formation location obtained from the wave generated by the large-diameter particle when the particle size changes, and... Figure 1 The right figure in the middle explains Figure 1 The left image in the middle and Figure 1 The droplet formation position in the combined wave state between the middle diagrams (specifically, the state obtained by the combined wave formed by one of the corresponding ones of b = a + b', c = a + c', and d = a + d'). Here, Figure 1 The 'a' in the figure describes the state of the BOP formed by the vibrating element in the absence of particles (the BOP changes with the machine). Figure 1 In the diagram, b indicates that the droplet fragmentation is the dominant state obtained by the vibrating element V. Figure 1 The 'c' in the equation indicates that the combination states are diverse and difficult to control, and Figure 1 The value 'd' indicates a state where fracture is dominated by waves of large-diameter particles. The fracture stability here is b > d >> c.

[0076] exist Figure 1In this context, the droplets obtained through the control of the driving voltage are known, but the waves and droplets induced by the propagation of large-diameter particles are typically not known. Therefore, the combined wave state of the two waves during large-diameter particle delivery is not known, and the droplet generation position of the former (the droplet obtained through the control of the driving voltage; see [link to relevant documentation]). Figure 1 The droplet generation location in (a) is different for each device, and the latter (the droplet generation location is determined by the propagation of large-diameter particles to the droplet wave; see also) Figure 1 The droplet generation position of the wave (b' to d') varies depending on the particle size, and therefore the aspects of performance degradation also vary.

[0077] In existing technical literature, large-diameter particles are uniformly treated by extending the charged region. On the other hand, in this technology, not only is the charged region extended, but the possibility of performance degradation is also determined by calculating ΔBOT or ΔBOP, and the performance degradation is mitigated by additionally determining parameters.

[0078] In other words, the main objective of this technology is to promote efficient sorting of large-diameter particles based on the combined state of two waves. Furthermore, this technology is a method for automatically determining sorting performance and setting parameters for performance improvement as needed. Therefore, the potential for a decline in sorting performance can be predicted without performing sorting, thus saving time and effort and improving the sorting performance itself. Additionally, the ability to predict a decline in sorting performance without performing sorting prevents sample waste.

[0079] The specific summary of this technology is as follows: the amplitude control of the vibrating element V, or the position of droplet formation by large-diameter particles, and the time elapsed from particle detection to reaching that position are calculated separately. For the two types of droplet formation, the difference between the positions where the droplet forms and breaks up, i.e., the break point (also known as "BOP"), is represented by ΔBOP. The difference in droplet formation time, i.e., the break time (also known as "BOT"), is represented by ΔBOT.

[0080] On the other hand, the "detection time" of the detection laser position and the particle at the detection laser position is considered as a reference. In the liquid column ejected from the orifice, the portion containing the particle is observed over time, and images of the waves (and droplets) appearing in the liquid column as the particle travels are acquired. The location of droplet formation or the time elapsed until droplet formation is calculated based on the acquired image set (however, the particle is not necessarily required for the calculation of BOP_1 described later). Until this point, the drive of the vibrating element V is turned off or set to low output drive.

[0081] In this technology, a decline in sorting performance can be predicted based on the value of ΔBOT or ΔBOP. When a decline in sorting performance is expected, additional adjustments to droplet control are performed. Specifically, when the two BOPs or BOTs are similar, or the latter's BOP is longer or its BOT is slower, the drive output of the vibrating element V is reduced, and then adjustments are performed to set the droplet as the sorting target. In the case of sorting droplets, the droplets are generated in the preceding liquid column portion, and the liquid column portion and the droplets break in the charged region, thus there is a possibility that they will be sorted. Therefore, when particles other than the sorting target are present between and within the piezoelectrically broken and particle-broken droplets, a function of not performing sorting can be provided. The range of particles other than the sorting target is preferably small, so the time or positional distance between the piezoelectrically broken and particle-broken droplets can be adjusted. At this time, the piezoelectrically broken droplets can be controlled so that the timing of the breakage becomes constant.

[0082] (3) Details of the particle sorting method involved in this embodiment

[0083] The particle sorting method involved in this embodiment includes at least the following steps: supplying a first driving voltage to a vibrating element V that applies vibration to a fluid; acquiring first optical information of the fluid and droplets generated by the vibration of the vibrating element V under the first driving voltage; supplying a second driving voltage lower than the first driving voltage to the vibrating element V; acquiring second optical information of the fluid and droplets containing second particles generated by the vibration of the vibrating element V under the second driving voltage; and determining parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

[0084] The deterioration in the sorting performance of large-diameter particles (hereinafter also referred to as "second particles") is due to the phenomenon that droplets, controlled by the drive of the vibrating element V to break at a constant time, become unstable due to the delivery of the large-diameter particles. In this technology, it has been found that this instability can be decomposed into two phenomena: a wave generated by the drive of the vibrating element V, and a wave generated independently of the aforementioned wave as the second particles travel. In this technology, by observing each wave and the breakage of the droplets generated by that wave, the presence or absence of a deterioration in sorting performance is predicted, and the setup for improving performance is implemented.

[0085] In the particle sorting method involved in this embodiment, the device settings can be based on either ΔBOT (break time) or ΔBOP (break point).

[0086] The details of the particle sorting method involved in this embodiment will be described below.

[0087] Adjustment using ΔBOT (break time)

[0088] The following section will describe a method for calculating ΔBOT and predicting the degradation of sorting performance.

[0089] ΔBOT = BOT_2 (hereinafter also referred to as "second BOT") - BOT_1 (hereinafter also referred to as "first BOT"), and ΔBOT can be obtained by measuring BOT_1 and BOT_2.

[0090] Figure 2 This is a diagram illustrating an example of the overall flow of the particle sorting method according to the present technology. In this embodiment, firstly, BOT_1 and BOT_2 are measured (S101, S102). Subsequently, ΔBOT is obtained, and if ΔBOT is below a threshold (S103), settings for BOT_2 are executed (S104). If ΔBOT exceeds the threshold, settings for BOT_1 are executed (S105).

[0091] The following will describe each step in detail.

[0092] The step of supplying a first driving voltage to the vibrating element V that applies vibration to the fluid.

[0093] Figure 3 This is a diagram illustrating an example of the measurement process for BOT_1. Figure 8 This is a diagram illustrating the image of BOT_1.

[0094] The vibrating element V vibrates the flow path at a predetermined frequency to apply minute vibrations to the sheath fluid, and the fluid (sample fluid and sheath fluid) exiting from the orifice is formed into droplets to generate a droplet flow. The vibrating element V is not particularly limited and can be appropriately and freely selected and used according to the purpose. Specifically, examples include piezoelectric vibrating elements V.

[0095] Vibration is applied to the fluid by supplying a first driving voltage to the vibrating element V, which is used to apply vibration to the fluid, and a first droplet suitable for sorting is formed (S201). In other words, by controlling the vibrating element V, a droplet suitable for sorting and having the shortest BOP is formed. The calibration bead can be used for the measurement of BOT_1.

[0096] The steps to obtain the first optical information of the fluid and droplets generated by the vibration of the vibrating element V under the first driving voltage.

[0097] In this step, the first optical information of the fluid and droplets generated by the vibration applied in the above steps is acquired by, for example, the optical information acquisition unit 15 described later. The optical information may be at least one of, for example, image and fluorescence information (e.g., fluorescence information of the sideflow).

[0098] This step further includes: detecting the particle by means of a calibration bead (hereinafter also referred to as "first particle") and by means of scattered light or fluorescence (S204), and based on the first optical information, setting the time from the reference time until the droplet containing the first particle breaks as a first BOT (BOT_1). In other words, BOT_1 is the time from particle detection to the breakage of the droplet containing the particle in a sortable droplet formed by the drive control of the vibrating element V. This can be obtained by calculating the elapsed clock when the first particle reaches the droplet generation section (S205).

[0099] In this embodiment, in the particle sorting system 1 described later, which is configured to detect scattered light or fluorescence generated by irradiating flowing particles with light, the timing of particle detection is set to a "reference time". The particle sorting system 1 described later includes an optical information acquisition unit 15, which can move in the direction of travel of the liquid column and observe the liquid column. It includes an LD or LED on the opposite side and has a light emission function that emits light at any time and at any emission time.

[0100] The step of supplying a second driving voltage lower than the first driving voltage to the vibrating element V

[0101] Figure 4 This is a diagram illustrating an example of the measurement process for BOT_2. Figure 9 This is a diagram illustrating the image of BOT_2.

[0102] In this step, a second driving voltage lower than the first driving voltage is supplied to the vibrating element V (including the case where the driving voltage is turned off) (S301). As the driving voltage, for example, a driving voltage of about 1% to 20%, preferably about 5% to about 15%, of the driving voltage during normal droplet control driving is supplied.

[0103] In this embodiment, the first optical information is optical information relating to a first particle (calibration bead) whose size is different from that of the second particle. In this embodiment, it is assumed that the second particle has a larger particle size than the first particle.

[0104] In this step, a sample liquid containing large-diameter particles is delivered (S302), and a second driving voltage is supplied to the vibrating element V that applies vibration to the fluid, thereby applying vibration to the fluid and forming a wave generated by the second particles (large-diameter particles).

[0105] The steps to acquire second optical information of fluid and droplets containing second particles generated by the vibration of the vibrating element V under the second driving voltage.

[0106] In this step, the second optical information of the fluid and droplets generated by the vibration applied in the above steps is acquired by, for example, the optical information acquisition unit 15 described later (S303). The optical information may be at least one of, for example, image and fluorescence information (e.g., fluorescence information of a sidestream). The droplets included in the second optical information are formed by waves generated by second particles (large-diameter particles).

[0107] The time until the droplet containing the second particle breaks apart is denoted as BOT_2. In other words, BOT_2 is the time from the detection of the droplet containing the particle breaking apart in a droplet induced by the particle's movement.

[0108] As a specific method for measuring BOT_2, the liquid column containing the second particle is observed (S304). In S304, n is any one of X, 1, and Y, where X and Y are arbitrary natural numbers. Subsequently, the imaging unit 151 (described later) images and observes the position where the particle arrives after n clock cycles from particle detection (S305). Subsequently, α images are acquired after n clock cycles from particle detection (S306). Subsequently, blurred images (β images) are removed, and the number of droplet-generated images (γ images) is counted (S307). Subsequently, the liquid column containing the particle is observed (S308).

[0109] This step further includes calculating the generation ratio of the droplet containing the second particle based on the second optical information. Specifically, as described above, optical information acquisition (in this case, image acquisition) of the liquid column portion containing the second particle is performed based on the elapsed time from the reference time. The elapsed time step is, for example, one clock unit driven by a piezoelectric motor. To acquire the optical information (preferably an image), the LED is strobe-activated at the instant of the elapsed time. The imaging unit 151 is moved to a position where the particle's arrival position can be observed at each elapsed time, multiple images are acquired at each elapsed time, and the generation ratio of the droplet is calculated. That is, here, the generation ratio of the droplet is calculated from the acquired image set.

[0110] This step further includes calculating a second BOT based on the calculated droplet generation ratio. That is, the droplet generation ratio is adjusted to an appropriate value by clock calculation to obtain BOT_2 (S309).

[0111] Here, BOT_1 is controlled to a constant value by the drive of the vibrating element V, but BOT_2 is a different value for each second particle. One of the main reasons for the variation is the size of the second particles. The size of the second particles varies substantially, so BOT is different for each second particle. In observations at earlier time points, not all particles have broken up (the droplet formation ratio is "0"). In observations at later time points, all particles have broken up (the droplet formation ratio is "1"). That is, the elapsed time at which the droplet formation ratio becomes appropriate can be called BOT_2 and used as an indicator. Examples of appropriate values ​​include the elapsed time calculated backward from the droplet formation ratio of relatively good sorting results (e.g., a droplet formation ratio of 0.5 ± 20%, preferably 0.5 ± 10%), but this embodiment is not limited to this.

[0112] As the BOT changes, the charged region containing all BOTs becomes longer, which leads to a decrease in purity (the proportion of sorted target particles contained in the sorted particles) and efficiency (the sorting efficiency of the sorted target particles). Therefore, this method is effective for large-diameter particles with small BOT changes, i.e., uniform second particle size, especially for carriers that hold cells in particles.

[0113] BOT_2 can be calculated based on the calculated droplet generation ratio, but it can also be calculated by applying a voltage to the liquid column for each elapsed time from the reference time to observe whether the charged droplets are deflected or not, and based on the presence or absence of the sorting target particles in the observed droplets. In this case, the driving voltage of the vibrating element V can be pre-off or set to a low output, and the driving voltage of the vibrating element V can be reset later. Alternatively, the above information acquisition can be performed while the driving voltage of the vibrating element V is gradually changed, and the driving voltage of the vibrating element V can be reset later.

[0114] In this step, image acquisition processing is performed only on the second particle (large-diameter particle) of the sorting target. This is achieved by judging and sorting target particles based on the intensity threshold of forward-scattered light, etc. For an image that is one of the optical information, if the integral of the stroboscopic emission of two or more particles occurs within the exposure time of the imaging unit 151 constituting the optical information acquisition unit 15, the image can be excluded from the determined objects.

[0115] The step of determining the parameters used to sort the second particles based on the comparison between the first optical information and the second optical information.

[0116] In this step, parameters for sorting the second particles are determined based on a comparison between the first optical information and the second optical information. Specifically, this step further includes predicting a decline in sorting performance based on a comparison between BOT_1 and BOT_2. More specifically, ΔBOT is obtained, and when its value meets a predetermined threshold (e.g., a clock value), the extent to which the sorting performance declines to below 80% is predicted. The degree of decline in sorting performance varies depending on the device configuration and particle size, and therefore cannot be uniquely determined. However, once the relationship between ΔBOT and performance decline is understood, the degree of performance decline can thereafter be estimated simply by measuring ΔBOT. The same applies to ΔBOP, which will be described later.

[0117] Subsequently, when ΔBOT meets a predetermined threshold, it is assumed that droplet breakage caused by the second particle is dominant, and the settings for BOT_2 are executed. On the other hand, when the threshold is not met, it is assumed that droplet breakage caused by the first particle is dominant, and the settings for BOT_1 are executed.

[0118] As stated above, from the viewpoint of fracture stability, BOT_1 is preferably constant. That is, this step preferably further includes controlling the parameters to make the first BOT constant.

[0119] The parameters may be any or more of the group consisting of, for example, sheath pressure, sample pressure, piezoelectric drive output, timing of charging the droplet, and charging range. In this specification, "sheath pressure" refers to the pressure applied to the sheath container, and "sample pressure" refers to the pressure applied to the sample tube.

[0120] Figure 5 This diagram illustrates an example of the setup process for BOT_1. According to the method described above, firstly, BOT_1 is measured (S401), and then the determined parameters are controlled to keep BOT_1 constant (S402). Subsequently, for example, steps of setting charged regions before and after the first BOT may be further included (S403).

[0121] Figure 6 This diagram illustrates an example of the setup process for BOT_2. According to the method described above, firstly, BOT_2 is measured (S501), and then the positional relationship between BOT_1 and BOT_2 is adjusted (S502). Subsequently, BOT_1 is controlled to be constant by controlling the determined parameters (S503). Then, for example, a step of setting a charged region before and after the second BOT may be further included (S504). This embodiment may further include a step of determining the charged region based on the generation ratio of droplets containing the second particle.

[0122] In the specific method for determining the parameters (here, the charged interval), the elapsed time BOT_1 is set as the center of the charged interval relative to the reference time. The range of the charged interval can be set to any range from "center - (M - 0.5) clocks" to "center + (N - 0.5) clocks" (M, N: natural numbers).

[0123] The method for determining the range of the charged region can be such that it includes the range where the droplet formation ratio X is 0 < X ​​< 1 within the elapsed time range searched to obtain BOT_2. Therefore, variations in BOT_2 can be covered.

[0124] The output of the drive of the vibrating element V can be changed to adjust ΔBOT within a range of ΔBOT ≥ 1 clock cycle. After adjustment, the output of the vibrating element V can be continuously fine-tuned until BOT_1 becomes a constant value. Therefore, by adjusting ΔBOT, the length of the liquid column portion that may be sorted even if it is not the sorting target can be controlled. The longer the liquid column portion, the higher the probability that particles other than the sorting target are present in the liquid column portion. When the breakup of the droplet formed by the drive of the vibrating element V occurs at the end of the charged region, the charge becomes incomplete, thus changing the flight distance. Therefore, by controlling BOT_1 to a constant value, droplet scattering can be prevented.

[0125] Furthermore, when it is known that particles other than the sorting target are present in the droplet or liquid column portion that breaks before BOT_2, it can be determined that sorting will not be performed. That is, the particle sorting method involved in this embodiment can further include the step of determining that sorting will not be performed when particles other than the sorting target are present in the liquid column region that breaks during the charged interval, in addition to the droplet that broke at the second BOT. Therefore, by determining that sorting will not be performed when particles other than the sorting target are present in the droplet and liquid column portion that breaks before BOT_2, purity can be improved. However, efficiency is reduced.

[0126] First variation

[0127] In this embodiment, the above adjustments can be combined with a method of repeatedly charging (continuously charging) droplets before and after the droplet of the target particle and sorting them together.

[0128] Second variation

[0129] When ΔBOT does not meet a predetermined threshold, or if ΔBOT is large, piezoelectrically controlled droplet breakage (BOT_1 becomes dominant) occurs before the wave accompanying the second particle's journey is generated and grown, thus reducing the likelihood of a decrease in sorting performance. Given that the droplet's BOT, obtained essentially by the control of the vibrating element V, is adjusted to its shortest value, the growth of the wave accompanying the particle's journey is largely independent of the device parameters. Therefore, there is a possibility of increasing ΔBOT by searching for droplet formation suitable for sorting under conditions where the device parameters are adjusted, such as by changing (e.g., reducing) the sheath pressure, to make BOT_1 breakage dominant. This method can mitigate the decline in sorting performance.

[0130] Third variation

[0131] Here, by using the fluorescence information of the sideflow as optical information, and by comparing the trajectory of the sideflow of the first particle with the trajectory of the sideflow of the large-diameter particle (the second particle), and adjusting each BOT so that the sideflow passes through an appropriate trajectory, efficient sorting can be achieved even when the target particles are large. In this variation, not only the sideflow but also the optical information from other parts can be used to achieve the above effect.

[0132] Adjustment using ΔBOP (breakpoint)

[0133] The following describes a method for calculating ΔBOP and predicting the decline in sorting performance.

[0134] By using this technique, similar to adjustments using ΔBOT, the potential for a decline in sorting performance can be predicted without performing sorting, thus saving time and effort and improving sorting performance.

[0135] ΔBOP = BOP_2 (hereinafter also referred to as "second BOP") - BOP_1 (hereinafter also referred to as "first BOP"), and ΔBOP can be obtained by measuring BOP_1 and BOP_2.

[0136] The step of supplying a first driving voltage to the vibrating element V that applies vibration to the fluid.

[0137] Figure 10 This is a diagram illustrating the image of BOP_1.

[0138] In this step, as in the adjustment using ΔBOT, the fluid is vibrated by supplying a first driving voltage to the vibrating element V, which vibrates the fluid, and a first droplet suitable for sorting is formed. In other words, by controlling the vibrating element V, droplets suitable for sorting and having the shortest BOT are formed.

[0139] In this embodiment, the calibration beads described above are not necessarily required for the measurement of BOP_1. This is because BOP_1 can be calculated without using the calibration beads.

[0140] The steps to obtain the first optical information of the fluid and droplets generated by the vibration of the vibrating element V under the first driving voltage.

[0141] In this step, the first optical information of the fluid and droplets generated by the vibration applied in the above steps is acquired by, for example, the optical information acquisition unit 15 described later. The optical information may be at least one of, for example, image and fluorescence information (e.g., fluorescence information of the sideflow).

[0142] This step further includes setting the location where the droplet breaks as the first break point (BOP) based on the first optical information. In other words, BOP_1 is the location where the droplet breaks based on the first optical information.

[0143] The step of supplying a second driving voltage lower than the first driving voltage to the vibrating element V

[0144] Figure 7 This is a diagram illustrating an example of the measurement process for BOP_2. Figure 11 This is a diagram illustrating the image of BOP_2.

[0145] In this step, a second driving voltage lower than the first driving voltage is supplied to the vibrating element V (including the case where the driving voltage is turned off) (S601).

[0146] In this embodiment, as in the adjustment using ΔBOT, the second particle is assumed to be a "large diameter particle". That is, BOP_2 is the location where the droplet containing the second particle (large diameter particle) breaks off.

[0147] In this step, a sample liquid containing large-diameter particles is delivered (S602), and a second driving voltage is supplied to the vibrating element V that applies vibration to the fluid, thereby applying vibration to the fluid and forming a wave generated by the second particles (large-diameter particles).

[0148] The steps to acquire second optical information of fluid and droplets containing second particles generated by the vibration of the vibrating element V under the second driving voltage.

[0149] In this step, the second optical information of the fluid and droplets generated by the vibration applied in the above steps is acquired by, for example, the optical information acquisition unit 15 described later (S603). The optical information may be at least one of, for example, image and fluorescence information (e.g., fluorescence information of a sidestream). The droplets included in the second optical information are formed by waves generated by second particles (large-diameter particles).

[0150] BOP_2 is the location where the droplet breaks off, containing the second particle (large diameter particle).

[0151] As a specific method for measuring BOP_2, firstly, the liquid column containing the second particle is observed (S604). In S604, n is any one of X, 1, and Y, where X and Y are arbitrary natural numbers. Subsequently, the imaging unit 151 (described later) images and observes the position reached by the particle after passing through n droplets from particle detection (S605). When using ΔBOP, the conversion can be performed using a length unit corresponding to one droplet cycle instead of a clock unit. The cycle length = flow rate × clock unit time holds true. Subsequently, α images are acquired after passing through n droplets from particle detection (S606). Subsequently, blurred images (β images) are removed, and the number of droplet-generated images (γ images) is counted (S607). Subsequently, the liquid column containing the particle is observed (S608).

[0152] This step further includes calculating the generation ratio of the droplet containing the second particle based on the second optical information. Specifically, as described above, optical information acquisition (in this case, image acquisition) of the liquid column portion containing the second particle is performed. The time step is, for example, a unit of length corresponding to one droplet cycle of the driving of the vibrating element V. To acquire optical information (preferably an image), the LED is strobe-activated at each elapsed time. The imaging unit 151 is moved to a position where the particle's arrival position can be observed at each elapsed time, multiple images are acquired at each elapsed time, and the droplet generation ratio is calculated. That is, here, the droplet generation ratio is calculated from the acquired image set.

[0153] This step further includes calculating a second BOP based on the calculated droplet generation ratio. That is, the position through which the droplet generation ratio is appropriately determined is calculated as BOP_2 (S609).

[0154] Here, BOP_1 is controlled to a constant value by the drive of the vibrating element V, but BOP_2 is a different value for each second particle. One of the main reasons for the variation is the size of the second particles. The size of the second particles varies substantially, so BOP is different for each second particle. In observations at earlier time points, not all particles have broken up (the droplet formation ratio is "0"). In observations at longer time points, all particles have broken up (the droplet formation ratio is "1"). That is to say, a BOP with a droplet formation ratio of 0.4 to 0.6 (preferably around 0.5) can be considered an average BOP_2 and is used as an indicator, but this embodiment is not limited to this numerical range.

[0155] As the BOP changes, the charged region encompassing all BOPs becomes longer, which leads to a decrease in purity and efficiency. Therefore, this method is effective for large-diameter particles with small BOP changes, i.e., uniform second particle size, especially for carriers in particles.

[0156] In this step, image acquisition processing is performed only on the second particle (large-diameter particle) of the sorting target. This is achieved by identifying target particles based on factors such as the intensity threshold of forward-scattered light. If, as one type of optical information, the image is excluded from the target selection if the integral of the stroboscopic emission of two or more particles occurs within the exposure time of the imaging unit 151 constituting the optical information acquisition unit 15, the image is considered as optical information.

[0157] The step of determining the parameters used to sort the second particles based on the comparison between the first optical information and the second optical information.

[0158] In this step, parameters for sorting the second particles are determined based on a comparison between the first optical information and the second optical information. Specifically, this step further includes a step of predicting a decrease in sorting performance based on a comparison between BOP_1 and BOP_2. More specifically, ΔBOP is obtained, and when its value meets a predetermined threshold (e.g., corresponding to the length of a droplet cycle), it is considered that droplet breakage obtained by the second particle is dominant, and settings for BOT_2 are executed. On the other hand, when the threshold is not met, it is considered that droplet breakage obtained by the first particle is dominant, and settings for BOT_1 are executed.

[0159] As stated above, from the viewpoint of fracture stability, BOP_1 is preferably constant. That is, this step preferably further includes the step of controlling the parameters to make the first BOP constant.

[0160] The parameters may be any one or more of the group consisting of, for example, sheath pressure, sample pressure, piezoelectrically driven output, timing of charging the droplet, and charging range.

[0161] The setup procedures for BOP_1 and BOP_2 can be obtained by replacing the setup procedures for BOT_1 and BOT_2 described above with those for BOP_1 and BOP_2, respectively.

[0162] 2. Second embodiment (particle sorting system 1)

[0163] Figure 12 This is a schematic conceptual diagram illustrating a first embodiment of the particle sorting system 1 according to the present technology, and Figure 13 This is a schematic conceptual diagram illustrating a second embodiment of the particle sorting system 1 according to the present technology.

[0164] The particle sorting system 1 according to this technology includes at least a vibrating element V, a voltage supply unit 13, an optical information acquisition unit 15, and a control unit 16. A flow path P, a light irradiation unit 11, a light detection unit 12, a vibrating element V, a sorting unit 14, an analysis unit 17, a storage unit 18, and a display unit 19 may be provided as needed.

[0165] Each unit will be described in detail below.

[0166] (1) Flow path P

[0167] The particle sorting system 1 according to this technology is a system for sorting particles flowing through a flow path P. The flow path P can be pre-set in the particle sorting system 1, or a commercially available flow path P, a disposable chip with a flow path P, etc., can be used.

[0168] There are no particular restrictions on the form of the flow path P, and it can be designed freely. For example, the flow path P is not limited to... Figure 12 The flow path P is formed in a two-dimensional or three-dimensional plastic (e.g., PP, PC, COP, or PDMS) or glass substrate T as shown in the first embodiment, and as Figure 13 The flow path P used in flow cytometers and the like in the related art, as shown in the second embodiment, can also be used in the particle sorting system 1 according to the present technology.

[0169] The width, depth, and cross-sectional shape of the flow path P are not particularly limited, as long as laminar flow can be formed, and can be freely designed. For example, microflow paths with a width of less than 1 mm can also be used in the particle sorting system 1 according to this technology. In particular, microflow paths with a width of about 10 μm or more and about 1 mm or less can be appropriately used in the particle sorting system 1 according to this technology.

[0170] In the flow path P, there are sample inlet P1 for introducing liquid (sample solution) containing particles that are the sorting target, and sheath inlet P2 for introducing sheath fluid. In the first embodiment, the sample solution is introduced into sample inlet P1 from sample solution storage section B1, merges with sheath fluid introduced into sheath inlet P2 from sheath fluid storage section B2, and is discharged from orifice P3 provided at the end of the flow path P.

[0171] A suction outlet P4 (not shown) for eliminating blockages or air bubbles can also be formed in the flow path P. A negative pressure source such as a vacuum pump can be connected to the suction outlet P4, and when blockages or air bubbles occur in the flow path P, the blockages and air bubbles can be eliminated by generating negative pressure in the flow path P and temporarily reversing the flow.

[0172] In this embodiment, "particles" can broadly include biologically related particles such as cells, microorganisms, and ribosomes, or synthetic particles such as latex particles, gel particles, and industrial particles. In this technology, particles are contained in fluids such as liquid samples.

[0173] Biologically relevant particles can include chromosomes, ribosomes, mitochondria, organelles (small organelles) that make up various cells. Cells can include animal cells (e.g., blood cells) and plant cells. Microorganisms can include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Biologically relevant particles can also include biologically relevant macromolecules such as nucleic acids, proteins, and their complexes.

[0174] Industrial particles can be, for example, organic or inorganic polymers, metals, etc. Examples of organic polymers include polystyrene, styrene-divinylbenzene, and polymethyl methacrylate. Examples of inorganic polymers include glass, silica, and magnetic materials. Examples of metals include colloidal gold and aluminum.

[0175] In this embodiment, the particles can be carriers or cells, and the carrier can hold one or more cells. For example, it can hold biological components (e.g., cells or cell-derived components (e.g., secretions)). Holding biological components in a carrier includes, for example, cases where the biological components are captured by the carrier and cases where the biological components are contained within the carrier. The carrier can be, for example, a carrier for secretion analysis. The carrier can be an emulsion, and in this case, the particles can be recovered in a state contained within the emulsion. In this case, the sorted product to be recovered can be an emulsion, and the dispersion medium constituting the emulsion can be particles contained within the emulsion containing the target particles for sorting. The dispersion medium constituting the emulsion can be appropriately selected by those skilled in the art based on, for example, the type of emulsion particles. The emulsion can be a multiple emulsion. Examples of multiple emulsions include, for example, oil-in-water-in-oil (“o / w / o”) or water-in-oil-in-water (“w / o / w”). The shape of the carrier is not particularly limited and can have any particle shape such as spherical, elliptical, or rod-shaped. Specific examples of carriers include the emulsions, beads, and gel capsules described above.

[0176] Particles flowing through flow path P can be labeled with one or more types of dyes, such as fluorescent dyes. 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), polydinophytin (PerCP), allophycocyanin (APC), 4',6-diamidinyl-2-phenylindole (DAPI), Cy3, Cy5, Cy7, and Brilliant Violet (BV421).

[0177] (2) Light irradiation unit 11

[0178] The particle sorting system 1 according to this embodiment may include a light irradiation unit 11. In the light irradiation unit 11, light is irradiated onto the particles flowing through the flow path P. In the particle sorting system 1 according to this embodiment, the light irradiation unit 11 is not necessary, and the particles flowing through the flow path P may also be irradiated with light by using an external light irradiation device or the like.

[0179] There are no particular restrictions on the type of light emitted from the light irradiation unit 11, but light with a constant light direction, wavelength, and intensity is preferred in order to reliably generate fluorescence or scattered light from the particles. Specific examples include lasers and LEDs. When using a laser, there are no particular restrictions on its type, and one or more types of argon (Ar) ion lasers, helium-neon (He-Ne) lasers, dye lasers, krypton (Cr) lasers, semiconductor lasers, solid-state lasers combining semiconductor lasers and wavelength conversion optical elements, etc., can be freely combined and used.

[0180] The particle sorting system 1 according to this embodiment can also employ so-called multi-spot illumination to illuminate multiple locations in the flow path P. In this case, multiple light illumination units 11 (not shown) can be provided, and light from one light illumination unit 11 is dispersed by a light control unit (not shown) such as a beam splitter, so that light can also illuminate multiple locations in the flow path P.

[0181] (3) Optical detection unit 12

[0182] The particle sorting system 1 according to this embodiment may include 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 the optical information emitted from particles flowing through the flow path P can also be detected by using an external light detection device or the like.

[0183] In the optical detection unit 12 that can be used in the particle sorting system 1 according to this embodiment, there are no particular limitations on the optical detection method, as long as the optical signal from the particles can be detected, and the optical detection method used in known optical detectors can be freely selected and used. Specifically, one or more optical detection methods used in fluorometers, scattering spectrometers, transmission spectrometers, reflection spectrometers, diffraction spectrometers, ultraviolet spectrometers, infrared spectrometers, Raman spectrometers, FRET meters, FISH meters, various other spectrometers, PMT arrays or photodiode arrays that arrange light receiving elements such as PMTs or photodiodes in one dimension, or devices that arrange multiple independent detection channels such as two-dimensional light receiving elements such as CCDs or CMOS are freely combined and used.

[0184] The particle sorting system 1 according to this embodiment can also perform light detection from multiple locations in the flow path P. In this case, although not shown, multiple light detection units 12 can be provided, or the light paths from multiple locations in the flow path P can be controlled by a light control unit (not shown) such as a reflector, so that one light detection unit 12 can also perform light detection from multiple locations in the flow path P.

[0185] In the particle sorting system 1 according to this embodiment, the arrangement of the optical detection unit 12 is not particularly limited as long as it can detect the optical signal from the particles, and can be designed appropriately and freely according to the purpose. For example, as Figure 1 and Figure 2 As shown in the first and second embodiments, the light detection unit 12 is preferably arranged on the opposite side of the light irradiation unit 11, with the flow path P sandwiched between them. By arranging the light detection unit 12 on the opposite side of the light irradiation unit 11 and sandwiching the flow path P, the light irradiation unit 11 and the light detection unit 12 can be arranged in a more flexible configuration. For example, since fluorescence is also emitted in a direction different from the incident direction of the irradiation light, the light detection unit 12 can be arranged on the same side as the light irradiation unit 11 or on a 90-degree side relative to the flow path P.

[0186] (4) Vibrating element V

[0187] The vibrating element V is as described in the "step of supplying a first driving voltage to the vibrating element V that applies vibration to the fluid" in "1. First embodiment (particle sorting method)".

[0188] The vibrating element V can contact the flow path P, and can be provided, for example, as an internal configuration of a substrate T including the flow path P, or as an internal configuration of the particle sorting system 1.

[0189] (5) Voltage supply unit 13

[0190] The voltage supply unit 13 supplies a driving voltage to the vibrating element V. The driving voltage of the vibrating element V is supplied according to a sine wave to form stable droplets, and is controlled by two parameters: frequency (clock value) and intensity (driving value). The specific control method is as described in "1. First Embodiment (Particle Sorting Method)" above, and therefore its description is omitted here.

[0191] (6) Sorting Unit 14

[0192] The particle sorting system 1 according to this embodiment may include a sorting unit 14 for performing particle sorting. The sorting unit 14 performs particle sorting based on data analyzed by the analysis unit 17 from the values ​​detected by the photodetector unit 12. For example, the sorting unit 14 may perform particle sorting on the downstream side of the flow path P based on analysis results such as particle size, morphology, and internal structure derived from the analysis data.

[0193] Specifically, by applying vibration to the entire or part of the flow path P using the aforementioned vibrating element V, droplets are generated from the outlet of the flow path P. By adjusting the amount of liquid fed into the flow path P, the diameter of the outlet, and the vibration frequency of the vibrating element V, the size of the droplets can be adjusted, and droplets containing a certain amount of microparticles can be generated.

[0194] Subsequently, based on the analysis results of the particle size, morphology, internal structure, etc., analyzed by the analysis unit 17, a positive or negative charge is applied to the droplet through a charging unit (not shown). The charging unit applies a positive or negative charge to the droplet discharged from the orifice P3 and includes a charged electrode, a voltage source for applying a predetermined voltage to the charged electrode, etc.

[0195] The charged electrode can be arranged to contact the sheath fluid and / or sample fluid flowing through the flow path P to apply a charge to the sheath fluid and / or sample fluid, and for example, a charged electrode inlet is provided in the substrate T including the flow path P, and the charged electrode can be inserted into the charged electrode inlet. The charged electrode can be arranged to contact the sample fluid, to contact the sheath fluid, or to contact both the sample fluid and the sheath fluid. However, considering the impact on the sorting of target particles (especially cells, etc.), the charged electrode is preferably arranged to contact the sheath fluid.

[0196] As described above, droplets containing any particles can be electrically separated by applying a positive or negative charge to the desired droplet. By synchronizing the charging timing of the charging unit with the voltage supplied to the vibrating element V, it becomes possible to charge only a specific droplet.

[0197] The charged droplets then change their path to a desired direction via deflection plates 141 and are sorted. Specifically, for example, deflection plates 141a and 141b change the direction of travel of each droplet in the fluid flow S by an electric current acting between the deflection plates 141a and 141b and the charge applied to the droplets, thereby guiding the droplets to a predetermined recovery container and arranging them such that the fluid flow S is sandwiched between them facing each other. As deflection plates 141a and 141b, conventionally used electrodes can be used, for example.

[0198] Different positive or negative voltages are applied to deflecting plates 141a and 141b, respectively. When charged droplets pass through the electric field formed by these voltages, an electric current (Coulomb force) is generated, and each droplet is attracted to one of the deflecting plates 141a and 141b. In particle sorting system 1, the direction of the flow (lateral flow) of droplets attracted by the electric field can be controlled by changing the positive / negative charge or the amount of charge on the droplets, thus allowing multiple particles that are different from each other to be sorted simultaneously.

[0199] Droplets whose trajectory is altered to the desired direction by deflection plates 141a and 141b are recovered by recovery containers 142a to 142c. General-purpose plastic tubing, glass tubing, etc., used in experiments can be used as recovery containers 142a to 142c. These recovery containers 142a to 142c are preferably arranged interchangeably in the system. The drain path of the recovered droplets can be connected to one of the recovery containers 142a to 142c that receives particles other than the sorting target.

[0200] There is no particular limitation on the number of recovery containers arranged in the particle sorting system 1. For example, in the case of more than three recovery containers, each droplet can be guided to and recovered into any one of the recovery containers depending on the presence or absence of the electric current between the deflection plates 141a and 141b and the magnitude of the electric force.

[0201] (7) Optical information acquisition unit 15

[0202] The optical information acquisition unit 15 acquires optical information of the fluid and droplets D discharged from the orifice P3 of the flow path P. The optical information may be at least one of, for example, image and fluorescence information (e.g., fluorescence information of the side flow).

[0203] The optical information acquisition unit 15 includes an imaging unit 151 for imaging a fluid or droplet D, a position adjustment mechanism 152 for adjusting the position of the imaging unit 151 to follow changes in the break point (BOP), and a data processing unit 153 for acquiring optical information of the fluid or droplet D from the image. As the imaging unit 151, in addition to imaging devices such as CCD cameras or CMOS cameras, various imaging elements such as photoelectric conversion elements can be used. According to this embodiment, the particle sorting system 1 can be equipped with a light source (not shown) that illuminates the imaging area together with the imaging unit 151.

[0204] The data processing unit 153 may include, for example, an information processing device comprising a general-purpose processor, a main storage device, and an auxiliary storage device. In this case, optical information can be obtained by inputting image data indicating the state of the fluid or droplet D imaged 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 disk, optical disk, magneto-optical disk, or flash memory, or may be distributed via wired or wireless networks.

[0205] (8) Control Unit 16

[0206] 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 includes, for example, an information processing device comprising a general-purpose processor, a main storage device, and an auxiliary storage device. The specific method for determining the parameters is as described in "1. First Embodiment (Particle Sorting Method)" above, and therefore its description is omitted here.

[0207] In the control unit 16, the parameters can be automatically controlled by inputting optical information acquired by the optical information acquisition unit 15 and executing a programmed control algorithm. Such a computer program can be stored on a recording medium such as a hard disk, optical disk, magneto-optical disk, or flash memory, or can be distributed via a network.

[0208] (9) Analysis Unit 17

[0209] The particle sorting system 1 according to this embodiment may further include an analysis unit 17 as needed. The analysis unit 17 is connected to the light detection unit 12 and analyzes the optical information detected by the light detection unit 12 from the microparticles.

[0210] The analysis unit 17 calculates characteristic quantities of each particle from optical information of light received, for example, from the light detection unit 12. Specifically, it calculates characteristic quantities indicating the particle's size, shape, internal structure, etc., from the detection values ​​of the received fluorescence or scattered light.

[0211] The analysis unit 17 is not essential in the particle sorting system 1 according to this embodiment, and the state of the particles can be analyzed 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 can be implemented by a personal computer or CPU, and can also be stored as a program in hardware resources including recording media (e.g., non-volatile memory (USB memory), HDD or CD), and made to function by a personal computer or CPU. The analysis unit 17 can be connected to each unit of the particle sorting system 1 via a network.

[0212] (10) Storage Unit 18

[0213] The particle sorting system 1 according to this embodiment may include a storage unit 18 for storing various types of information. The storage unit 18 may store information data detected by each optical 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, data on particles sorted by the sorting unit 14, and all other information.

[0214] In the particle sorting system 1 according to this embodiment, the storage unit 18 is not necessary, and an external storage device can be connected. For example, a hard disk can be used as the storage unit 18.

[0215] (11) Display unit 19

[0216] The particle sorting system 1 according to this embodiment may include a display unit 19 for displaying various information. The display unit 19 may display information data detected by each optical 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, data of particles sorted by the sorting unit 14, and all other information.

[0217] In the particle sorting system 1 according to this embodiment, the display unit 19 is not necessary, and an external display device can be connected. For example, a monitor and a printer can be used as the display unit 19.

[0218] (12) Other

[0219] In the particle sorting system 1 according to this embodiment, the flow path P, the light irradiation unit 11, the light detection unit 12, the vibration element V, the optical information acquisition unit 15, the analysis unit 17, the storage unit 18, the display unit 19, etc., can be arranged independently as needed. For example, the flow path P can be pre-set in the particle sorting system 1, or a commercially available flow path P, a disposable chip with the flow path P, etc., can be installed in the particle sorting system 1 to perform analysis or sorting.

[0220] The light irradiation unit 11 and the light detection unit 12 can be pre-installed in the particle sorting system 1, but the light irradiation of particles flowing through the flow path P and the light detection of particles can also be performed by using an external light irradiation device, an external light detection device, etc. The optical information acquisition unit 15, the analysis unit 17, the storage unit 18, the display unit 19, etc., can be pre-installed in the particle sorting system 1, or external detection devices, external analysis devices, external storage devices, external display devices, etc., can also be used. In this case, the various devices can also be connected to each other via wired or wireless networks.

[0221] 3. Third Implementation Example (Program)

[0222] The program according to this embodiment is a program that enables 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 the above-described "2. Second Embodiment (Particle Sorting System 1)". This program can also be distributed independently.

[0223] The program according to this embodiment can be provided stored in a recording medium such as a disk, optical disk, magneto-optical disk, or flash memory, and can be downloaded to a computer and used. Alternatively, the program according to this embodiment distributed externally can be downloaded to a computer and used via a network such as the Internet.

[0224] A computer that has downloaded the program according to this embodiment acquires information about ΔBOT or ΔBOP and executes the control algorithm of the downloaded program to determine the parameters. Based on the determined parameters, the computer issues instructions to the particle sorting system 1, and the parameters of the particle sorting system 1 are automatically controlled.

[0225] Note that this technology can also be configured as follows. (1)

[0227] A particle sorting method, comprising: The step of supplying a first driving voltage to a vibrating element that applies vibration to a fluid. The step of acquiring first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage. The step of supplying the vibration element with a second driving voltage lower than the first driving voltage. The step of acquiring second optical information of fluid and droplets containing second particles generated by the vibration of the vibrating element under the second driving voltage, and The step of determining the parameters for sorting the second particles based on a comparison between the first optical information and the second optical information. (2)

[0229] According to the particle sorting method described in (1), the optical information is at least one of image and fluorescence information. (3)

[0231] According to the particle sorting method described in (1) or (2), the droplets contained in the second optical information are formed by waves generated by the second particles. (4)

[0233] According to the particle sorting method described in (1), the first optical information is related to the first particle, which has a size different from that of the second particle. (5)

[0235] According to the particle sorting method described in (4), the second particle has a larger particle size than the first particle. (6)

[0237] The particle sorting method according to (4) or (5) further includes the step of setting a first breakage time (BOT) based on the first optical information, up to the time from the reference time until the droplet containing the first particle breaks. (7)

[0239] The particle sorting method according to (6) further includes the step of calculating the generation ratio of droplets containing the second particles based on the second optical information. (8)

[0241] The particle sorting method according to (7) further includes the step of applying a voltage to the liquid column for the calculated generation ratio of the droplets or for each elapsed time from the reference time, to observe whether the charged droplets are deflected or not, and calculating a second BOT based on the presence or absence of the target particles in the observed droplets. (9)

[0243] The particle sorting method according to (8) further includes a step of predicting the decline in sorting performance based on a comparison between the first BOT and the second BOT. (10)

[0245] The particle sorting method according to (9) further includes the step of controlling the parameters to make the first BOT constant. (11)

[0247] According to the particle sorting method described in (10), the parameter is any one or more of the group consisting of sheath pressure, sample pressure, piezoelectric drive output, timing of charging the droplet, and charging interval. (12)

[0249] According to the particle sorting method described in (11), the method further includes the step of setting the charged interval before and after the first BOT. (13)

[0251] The particle sorting method according to any one of (1) to (12) further includes the step of determining the charged region based on the generation ratio of droplets containing the second particle. (14)

[0253] The particle sorting method according to (11) further includes a step of determining not to perform sorting when, in the region of the liquid column that broke during the charged interval, there are particles other than the sorting target, in addition to the droplets that broke at the second BOT. (15)

[0255] The particle sorting method according to (1) further includes the step of setting the position of the droplet breakage as the first break point (BOP) based on the first optical information. (16)

[0257] The particle sorting method according to (15) further includes the step of calculating the generation ratio of droplets containing the second particles based on the second optical information. (17)

[0259] The particle sorting method described in (16) further includes the step of calculating a second BOP based on the calculated generation ratio of the droplets. (18)

[0261] The particle sorting method according to (17) further includes a step of predicting the decline in sorting performance based on a comparison between the first BOP and the second BOP. (19)

[0263] A particle sorting system, comprising: The voltage supply unit supplies a first driving voltage to the vibrating element that applies vibration to the fluid; and The optical information acquisition unit acquires first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage, wherein... The voltage supply unit supplies a second driving voltage, lower than the first driving voltage, to the vibrating element, and The optical information acquisition unit acquires second optical information of the fluid and droplets containing the second particles generated by the vibration of the vibrating element under the second driving voltage, and The particle sorting system further includes a control unit, which determines parameters for sorting the second particles based on a comparison between the first optical information and the second optical information. (20)

[0265] A program that executes: A first driving voltage is supplied to the vibrating element that applies vibration to the fluid. Acquire first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage. A second driving voltage lower than the first driving voltage is supplied to the vibration element. Acquire second optical information about the fluid and droplets containing the second particles generated by the vibration of the vibrating element under the second driving voltage, and Based on the comparison between the first optical information and the second optical information, the parameters used to sort the second particles are determined.

[0266] Example

[0267] The present invention will now be described in more detail with reference to examples. Note that the examples described below are representative examples of the present invention, and the scope of the present invention should not be narrowly interpreted in light of these examples.

[0268] Example 1: Using ΔBOT's automatic adjustment

[0269] Automatic adjustment for calculating ΔBOP and predicting sorting performance degradation

[0270] summary

[0271] In this example, the presence or absence of a decrease in sorting performance can be predicted to some extent by the value of ΔBOT. In droplet-based cell sorters, the droplet is typically controlled to make BOT_1 as short as possible. However, for example, if the clock value of the vibrating element V under ΔBOT is below a predetermined threshold, there is a possibility that interference between the two waves occurs and the droplet breaks down, becoming unstable. Furthermore, the yield decreases simply by applying charge in the time interval before and after BOT_1. Alternatively, if BOT_2 is shorter than BOT_1, the usual charge application based on BOT_1 cannot apply charge to droplets containing the second particle (large-diameter particle), and the droplet is not sorted (because the droplet has already broken down at the BOT_2 time point).

[0272] The threshold is set appropriately based on the cell sorter, the orifice diameter used, and the particle size. The threshold is based on the time interval from BOT_2 back to the point at which the wave generated by the particle's propagation is first observed.

[0273] (Adjusted example)

[0274] In this example, a particle sorting system 1 is used, configured to detect scattered light or fluorescence generated by irradiating flowing particles with a laser. The timing of particle detection is referred to as the "reference time". The particle sorting system 1 includes an optical information acquisition unit 15, which can move in the direction of travel of the liquid column and observe the liquid column. The particle sorting system 1 includes an LD or LED on opposite sides and has a light-emitting function that emits light at any time and at any emission time.

[0275] Subsequently, by controlling the vibrating element V, droplets suitable for sorting and with the shortest BOP are formed. The time from the reference time until the droplet containing the first particle breaks apart is denoted as BOT_1 (see...). Figure 8 In this example, the calibration bead is used for the measurement of BOT_1.

[0276] Subsequently, the output of vibrating element V is turned off or set to a low output, and the output of vibrating element V is adjusted by a low output drive to the extent that droplet formation occurs through vibrating element V. When the output of vibrating element V is turned off, droplet formation obtained by natural jetting dominates. At this time, there is a possibility of droplet scattering and contamination inside the device, so it is preferable to perform a minimum drive, such that droplet formation driven by vibrating element V dominates. However, if the possibility of droplet scattering and contamination inside the device does not need to be considered, the output of vibrating element V can be turned off.

[0277] The output of the vibrating element V is supplied with a driving voltage of approximately 1% to 20%, preferably approximately 5% to 15%, of the driving voltage used in normal droplet control driving. The time until the droplet containing the second particle (large-diameter particle) breaks apart, relative to the reference time, is then denoted as BOT_2 (see [reference]). Figure 9 ).

[0278] As a specific method for measuring BOT_2, the imaging unit 151 is moved to the liquid column containing the second particle based on the elapsed time from the reference time, and observation is performed (see...). Figure 14 At this point, when the phase of the clock at the detection time of the second particle is denoted as Φx, the range of Φx can be limited, and the liquid column can be observed only for the second particle detected at a phase within a specified range (e.g., a range of 20% of the phase in the center of a wavelength, preferably a range of 10% of the phase in the center of a wavelength). In liquid column observation, the position of the second particle changes by one wavelength; therefore, by limiting the phase, the change in the observed position of the particle is suppressed, and each particle is easily captured at the center of the observed image.

[0279] The time step is, for example, one clock unit for driving the vibrating element V. To acquire an image, an LED arranged opposite the optical information acquisition unit 15 emits a radio flash immediately after the time has elapsed. The location of the second particle at each elapsed time is predicted, and the imaging unit 151 needs to be moved appropriately so that the wave or droplet generated by the particle is captured in the image. Multiple liquid column images are acquired at each elapsed time, and the droplet generation ratio is calculated (see...). Figure 15 The experimental conditions here are as follows: the device is a cell sorter (SH800: manufactured by Sony), the chip is a 130μm M1 chip (manufactured by Sony), the sample is 40μm beads, and the mode is set to 3 drops per run. An approximate straight line is plotted on the relationship between elapsed time and the generation ratio, and based on this approximate straight line, the elapsed time for the generation ratio of droplets is calculated and denoted as BOT_2. The generation of droplets can be determined, for example, by image processing to determine how many clusters of liquid portions (displayed as black portions in the image) with a certain area are present in the image (see [link to relevant documentation]). Figure 16 ).

[0280] Subsequently, the second particle (large-diameter particle) of the sorting target is gated based on forward scattered light (FSC), backscattered light (BSC), side scattered light (SSC), fluorescence, etc. Optical information (in this case, an image) of the liquid column containing the second particle is obtained using the gated particle as the target.

[0281] During image acquisition, when the integral of the flickering emission of two or more particles occurs within the exposure time of the imaging unit 151, the image becomes blurred (see...). Figure 17 Therefore, this image is excluded from the calculation of the droplet generation ratio. Such an image is randomly generated based on the imaging frame rate and the number of events per second (EPS) of particles (the particle arrival interval is theoretically considered to be events according to a Poisson process).

[0282] The choice between capturing an image of a single particle and capturing images of multiple particles can be determined based on, for example, the average brightness value of each image (see...). Figure 18 For each acquired image, an average brightness value is calculated, and the image near its minimum value is the one that captured a single flicker (one particle). Therefore, a value ranging from the minimum value to the coefficient is selected. Images within the range of the Min value are used as a reference for calculating the droplet generation ratio. Bright images outside the range are considered as images capturing multiple strobes (multiple particles) and are excluded from the determination of the droplet generation ratio.

[0283] Then, ΔBOT = BOT_2 - BOT_1 is calculated. For example, in this example, when ΔBOT is below 7.2 clock cycles, the predicted degradation in sorting performance is (see...). Figure 19 ). Figure 19 This is a plot of the recovery rate against ΔBOT for a 30μm bead. It can be seen that the recovery rate begins to decrease when ΔBOT drops below approximately 0.6 ms. At this point, the piezoelectric element's clock is 12kHz, so 0.6 ms corresponds to 7.2 clock cycles. In this example, 7.2 clock cycles are set as a predetermined threshold for the vibrating element V under ΔBOT, but this is only an example.

[0284] Automatic adjustment for setting particle-trajectorized droplets as sorting targets

[0285] summary

[0286] In this example, droplets induced by particle travel can be set as sorting targets. Here, when the interference of the two waves is the cause of the degraded sorting performance, it is preferable to create a state where one wave dominates. Given that BOT_1 has been controlled close to its minimum, BOT_2 is not a phenomenon primarily dependent on device control; therefore, there is no obvious means to make BOT_1 dominate in the state of interference between the two waves. Therefore, to create a state where BOT_2 dominates, the drive output of the vibrating element V is intentionally reduced, and BOT_1 is lengthened. As BOT_2 becomes shorter than BOT_1, BOT_2 becomes more dominant at the breakage point. In the state where BOT_2 dominates, droplets induced by particle travel can be set as sorting targets by applying charge in the time intervals before and after BOT_2.

[0287] Adjustment Example

[0288] Perform the "automatic adjustment for calculating ΔBOP and predicting the decline in sorting performance" process. Subsequently, for each particle, the range of the charged interval can be set as an arbitrary range from "center - (M - 0.5) clocks" to "center + (N - 0.5) clocks" (M, N: natural numbers) based on the timing when the elapsed time from the reference time becomes BOT_2.

[0289] In a specific method for determining the length of the charged region, the charged region can be defined such that it includes a range in which the droplet generation ratio X is 0 < X ​​< 1 within the elapsed time range searched to obtain BOT_2. The width of this range increases with the size variation of the target particles being sorted, and a wider range leads to a decrease in efficiency. The greater the variation in the breakup location, the more likely the droplet's flight distance will change during sorting; therefore, a small variation is preferable. Thus, the method of setting the droplets generated along with the particle's journey as the sorting target is particularly effective for targeting carrier particles with uniform particle size.

[0290] The drive output of the vibrating element V can be changed to adjust ΔBOT within the range of "ΔBOT ≥ 1 clock cycle". After adjustment, the piezoelectric output can be continuously fine-tuned to make BOT_1 a constant value (by properly controlling BOT_1, the large-scale scattering of droplets breaking at the position BOP_1 can be suppressed). Charging the liquid column requires a certain amount of time during its rise and fall, and the charge of the broken droplets becomes incomplete during this interval, leading to changes in the droplet's landing position unless proper control is implemented.

[0291] Droplets that break off before BOT_2 during the charging of the liquid column may be sorted together with droplets that break off at BOT_2. Therefore, when particles other than the sorting target are present in the region of the liquid column that broke off during charging, besides the droplets that broke off at BOT_2, the control unit 16 can determine not to perform sorting (see [link]). Figure 20 ).

[0292] Example 2: Using ΔBOP auto-adjustment

[0293] summary

[0294] In the first example above, ΔBOT was used to perform automatic adjustment, but in this technique, ΔBOP can be used instead of ΔBOT.

[0295] Adjustment Example

[0296] Calculate ΔBOP = BOP_2 - BOP_1.

[0297] In the case of ΔBOT, the prediction of sorting performance degradation is determined in clock units, but in the case of ΔBOP, ΔBOP can be obtained by converting it to a length unit corresponding to one droplet cycle.

[0298] Example 3: Using multiple drops to set the charged range

[0299] summary

[0300] Figure 21This is a diagram illustrating the concept of multiple drops. The "multiple drops" described in this specification refers to the function of changing the number of charged droplets before and after the optimal delay determined by calibration. In Example 3, by performing multiple drop adjustment, even with the optimal delay timing for the first particle, the charged interval is extended until the BOP (Breakpoint of the second particle) is included, and it can cover changes in the break position (or make the total charge of the droplets merging during flight more uniform), thus improving the recovery rate of the second particle (large-diameter particle). That is, even without observing the second particle, when the recovery rate of sorting the second particle under the determined conditions of the first particle is poor, or when afterimages (jittering) are observed by observing the droplet state during the delivery of the second particle, the recovery rate of the second particle can be improved by increasing the charged interval (the number of charged droplets). However, there is a problem that the number of aborts also increases with the number of charged droplets, therefore, the charged interval needs to be set appropriately.

[0301] Adjustment Example

[0302] Figures 22 to 24 , Figure 26 and Figure 28 These are diagrams illustrating the actual execution of multiple drops. Figures 22 to 24 , Figure 26 and Figure 28 In the diagram, "column" represents the "liquid column," "particle" represents the "second particle," and "droplet" represents the "liquid droplet." The horizontal axis represents the number of clock units, and the vertical axis represents the number of droplet cycles. The area within the thick box in each figure represents the monitoring target. Figures 22 to 24 , Figure 26 and Figure 28 In the diagram, the charged second particle and the droplet are represented by being surrounded by circles.

[0303] Figure 22 This is a diagram illustrating the sorting process performed in a single-drop charged mode. Figure 23 This is a diagram illustrating the sorting process performed in the three-drop charged mode. Figure 22 and Figure 23 An example representing the case of normal particles that do not undergo critical fracture is that the charging timing is preferably near the time when the target particle arrives at the BOP.

[0304] Figure 24 This represents the case of a three-drop charging mode with a critical fracture (Pa) of 15, a piezoelectric element (PZT) fracture (Pi) of 18, and ΔBOP (N) = 3 in low-drive operation. Following the same approach, when the three-drop charging mode is set with the critical fracture as the charging reference, as shown... Figure 25 As shown, there is a possibility of charged droplets forming outside the monitoring area (outside the thick box) (i.e., a risk of contamination). In low-drive sorting with priority efficiency, three charged droplets are used as a baseline based on BOT_2. Specifically, the method described above can be used... Figure 6 The flowchart shown illustrates the method for setting the charged region.

[0305] Figure 26 This represents the case of a three-droplet charged mode with a critical fracture (Pa) of 15, a piezoelectric element (PZT) fracture (Pi) of 18, and ΔBOP(N) = 3 in low-drive actuation. In other words, to avoid the above... Figure 25 The state shown allows for setting the charged region to cover the critical fracture (Pa) by multiple drops while maintaining the PZT fracture (Pi) at 18. Therefore, ideal charging can be performed when observing the BOP (Pa) of the second particle and covering the critical fracture (Pa) by multiple drops. In low-drive sorting with priority purity, BOT_1 (however, this is in...) Figure 2 The value obtained at time point S101 is used as a reference to include BOT_2 using multiple charges. Specifically, as Figure 27 As shown in the flowchart, in S701 to S703, the above-described... Figure 6 The flowchart shown illustrates methods S501 to S503, and in S704, it is possible to... Figure 2 Based on BOT_1 obtained in S101, a prior charged range is set to include BOT_2.

[0306] Figure 28 This specifically refers to the case where the particle velocity is relatively low. In this situation, the particle is caught up with and collides with a subsequent droplet during its flight, thus the collision effectively charges the subsequent droplet. Therefore, in Figure 28 In the case shown, an eight-drop charged mode is used. Specifically, when the particle velocity of the second particle (large diameter particle) is relatively low, a low-drive setting is not executed when BOT_2 is obtained, and instead BOT_1 is used (however, this is in...). Figure 2 The time point obtained at S101 is used as a reference to use multiple charges to include BOT_2, and also includes subsequent intervals. Specifically, as Figure 29 As shown in the flowchart, in S801, the method according to the above... Figure 6 The method in S501 of the flowchart shown can be implemented in S802 as follows: Figure 2 The BOT_1 obtained in S101 is used as a reference to set a prior charged region to include BOT_2, and the charged region can be set so that subsequent charged regions also include multiple droplets.

[0307] Experimental Example

[0308] Figure 30This is a graph showing the results when using multi-drop sorting for the second particle. The vertical axis represents Recovery, and the horizontal axis represents Current (sorting in three-drop charged mode) and New (sorting with extensive multi-drop processing before and after; performing the above...). Figure 28 (The example shows the sorting method). In this example, the second particle sorted is a large-diameter particle with a diameter of 50 μm to 70 μm. The results from this example demonstrate that by using multiple drops to define the charged region, the recovery rate of large-diameter particles is improved.

[0309] List of reference numerals

[0310] 1. Particle sorting system

[0311] P flow path

[0312] P1 Sample Entry

[0313] P2 Sheath Inlet

[0314] P3 orifice

[0315] 11 Light Illumination Units

[0316] 12 optical detection units

[0317] V Vibrating element

[0318] 13 Voltage Supply Unit

[0319] 14 sorting units

[0320] 141a, 141b deflection plates

[0321] 142a to 142c Recycling Containers

[0322] 15 Optical Information Acquisition Unit

[0323] 151 Imaging Unit

[0324] 152 Position Adjustment Mechanism

[0325] 153 Image Data Processing Unit

[0326] BOT break time

[0327] BOP break point

[0328] 16 Control Unit

[0329] D droplets

[0330] 17 Analysis Unit

[0331] 18 Storage Department

[0332] 19 Display Section

Claims

1. A particle sorting method, comprising: A first driving voltage is supplied to a vibrating element configured to apply vibration to a fluid; Acquire first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage; A second driving voltage lower than the first driving voltage is supplied to the vibration element; Acquire second optical information of the fluid and droplets containing the second particles generated by the vibration of the vibrating element under the second driving voltage; as well as Based on the comparison between the first optical information and the second optical information, the parameters used to sort the second particles are determined.

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

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

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

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

6. The particle sorting method according to claim 4, further comprising: Based on the first optical information, the time until the droplet containing the first particle breaks apart relative to the reference time is set as the first breakage time BOT.

7. The particle sorting method according to claim 6, further comprising: Based on the second optical information, the generation ratio of droplets containing the second particle is calculated.

8. The particle sorting method according to claim 7, further comprising: A voltage is applied to the liquid column for the calculated droplet generation ratio, or for each elapsed time from the reference time, to observe whether the charged droplets are deflected or not, and a second BOT is calculated based on the presence or absence of the sorted target particles in the observed droplets.

9. The particle sorting method according to claim 8, further comprising: Based on the comparison between the first BOT and the second BOT, the decline in sorting performance is predicted.

10. The particle sorting method according to claim 9, further comprising: The parameters are controlled to keep the first BOT constant.

11. The particle sorting method according to claim 10, wherein, The parameters are any one or more of the following groups: sheath pressure, sample pressure, piezoelectric drive output, timing of charging the droplet, and charging range.

12. The particle sorting method according to claim 11, further comprising: The charged range is set before and after the first BOT.

13. The particle sorting method according to claim 1, further comprising: The charged region is determined based on the generation ratio of droplets containing the second particle.

14. The particle sorting method according to claim 11, further comprising: If, in the region of the liquid column that breaks during the charged interval, there are particles other than the sorting target, in addition to the droplet that breaks at the second BOT, it is determined that sorting will not be performed.

15. The particle sorting method according to claim 1, further comprising: Based on the first optical information, the location where the droplet breaks is set as the first break point BOP.

16. The particle sorting method according to claim 15, further comprising: Based on the second optical information, the generation ratio of droplets containing the second particle is calculated.

17. The particle sorting method according to claim 16, further comprising: The second BOP is calculated based on the calculated droplet generation ratio.

18. The particle sorting method according to claim 17, further comprising: Based on the comparison between the first BOP and the second BOP, the decline in sorting performance is predicted.

19. A particle sorting system, comprising: A voltage supply unit is configured to supply a first driving voltage to a vibrating element configured to apply vibration to a fluid; as well as The optical information acquisition unit is configured to acquire first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage, wherein... The voltage supply unit supplies a second driving voltage, lower than the first driving voltage, to the vibrating element, and The optical information acquisition unit acquires second optical information of the fluid and droplets containing the second particles generated by the vibration of the vibrating element under the second driving voltage, and The particle sorting system further includes a control unit configured to determine parameters for sorting the second particles based on a comparison between the first optical information and the second optical information.

20. A program configured to perform the following operations: A first driving voltage is supplied to a vibrating element configured to apply vibration to a fluid; Acquire first optical information about the fluid and droplets generated by the vibration of the vibrating element under the first driving voltage; A second driving voltage lower than the first driving voltage is supplied to the vibration element; Acquire second optical information of the fluid and droplets containing the second particles generated by the vibration of the vibrating element under the second driving voltage; as well as Based on the comparison between the first optical information and the second optical information, the parameters used to sort the second particles are determined.

Images