Particle separation device, particle separation method, and computer-readable storage medium

Abstract

A particle separation apparatus, a particle separation method, and a computer-readable storage medium are provided. The provided particle sorting apparatus includes a vibration unit that applies vibration to a fluid containing a sheath fluid by providing a driving voltage based on a plurality of frequencies; an imaging unit that acquires an image of the fluid and a droplet at a position where the fluid is converted into the droplet by the vibration; a liquid temperature control unit that controls a liquid temperature of the sheath fluid; and a frequency control unit that, for each liquid temperature of the sheath fluid, acquires data related to break-off of the droplet at each frequency from an image taken by the imaging unit, and controls the frequency of the driving voltage based on a variation in the data, which variation accompanies a change in the liquid temperature of the sheath fluid.

Description

Technical Field

[0001] This technology relates to particle separation apparatus, particle separation method, and procedure. More specifically, this invention relates to a particle separation apparatus, particle separation method, and procedure capable of stabilizing droplets. Background Technology

[0002] Currently, a technique called flow cytometry is used to analyze biologically relevant particles (such as cells and microorganisms) as well as microbeads. Flow cytometry is an analytical method in which particles are poured in a fluid-aligned state and illuminated with light to detect the light emitted from each particle, thereby analyzing and separating the particles. The device used for flow cytometry is called a flow cytometer (also known as a "cell sorter").

[0003] In flow cytometry, a vibrating element is typically positioned in the section of the flow path through which particles encased in sheath fluid pass. This vibrating element vibrates a portion of the flow path to continuously form droplets from the outlet. Then, based on a detection signal obtained through light irradiation, the droplets containing particles are either positively (+) or negatively (-), or remain uncharged, and split according to their charge state by a deflection plate. The target particles are collected in appropriate recovery containers. The groups of droplets deflected to the left or right by the positive or negative charge follow a specific trajectory, appearing as a linear, inclined liquid flow. The vertically downward-moving group of uncharged droplets is called the "center flow," while the inclined linear liquid flow is called the "side flow."

[0004] Stabilizing the droplets is important so that the lateral flow is properly guided to the recovery container to maintain a constant lateral flow trajectory. In response, for example, Patent Document 1 discloses a technique for stabilizing droplets by controlling the driving voltage of a vibrating element, such that the distance between the tip of the droplet just before it breaks off and the tip of an adjoint droplet at a previous position is constant in the droplet observation image. Furthermore, Patent Document 2 discloses a technique for stabilizing droplets by controlling the driving voltage of the vibrating element by focusing on the length of the first adjoint. Additionally, Patent Document 3 discloses a technique for controlling the liquid temperature of the sheath fluid in a sheath fluid introduction unit that introduces sheath fluid into the flow path to prevent droplet instability caused by variations in the flow rate of the sheath fluid due to changes in ambient temperature, although the primary purpose is to stabilize the flow rate during measurement.

[0005] Reference List

[0006] Patent documents

[0007] Patent Document 1: WO 2014 / 115409 A

[0008] Patent Document 2: Japanese Patent Application Publication No. 2006-292769

[0009] Patent Document 3: Japanese Patent Application Publication No. 2019-190991 Summary of the Invention

[0010] The problem to be solved by the present invention

[0011] However, the reality is that the technology used to maintain a constant lateral flow trajectory and stabilize droplets is still insufficient and needs further development.

[0012] Therefore, the main purpose of this technology is to provide a technique that can stabilize droplets.

[0013] Solution to the problem

[0014] This technology provides a particle separation device, including: a vibration unit that applies vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies; an imaging unit that acquires images of the fluid and each droplet at locations where the fluid is formed into droplets by vibration; a liquid temperature control unit that controls the liquid temperature of the sheath fluid; and a frequency control unit that acquires data on the state of the droplets at each frequency at each liquid temperature of the sheath fluid from images captured by the imaging unit, and controls the frequency of the driving voltage based on variations in the data accompanying changes in the liquid temperature of the sheath fluid.

[0015] Furthermore, this technology also provides a particle separation method, comprising: a vibration step, applying vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies; an imaging step, acquiring images of the fluid and each droplet at locations where the fluid is formed into droplets by vibration; a liquid temperature control step, controlling the liquid temperature of the sheath fluid; and a frequency control step, acquiring data on the state of the droplets at each frequency for each liquid temperature of the sheath fluid from the images acquired in the imaging step, and controlling the frequency of the driving voltage based on variations in the data accompanying changes in the liquid temperature of the sheath fluid.

[0016] Furthermore, this technology also provides a program for causing a particle separation device to perform the following functions: applying vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies; acquiring images of the fluid and each droplet at locations where the fluid is formed into droplets by vibration; controlling the liquid temperature of the sheath fluid; and acquiring data from the captured images regarding the state of the droplets at each frequency at each liquid temperature of the sheath fluid, and controlling the frequency of the driving voltage based on variations in the data accompanying changes in the liquid temperature of the sheath fluid. Attached Figure Description

[0017] Figure 1This is a diagram illustrating a configuration example of a first embodiment of a particle separation apparatus according to the present technology.

[0018] Figure 2 This is a diagram illustrating another configuration example of a particle separation apparatus according to the present technology, representing a first embodiment.

[0019] Figure 3 This is a diagram showing the relationship between a droplet cycle and the correct timing of the charging signal.

[0020] Figure 4 It is a view showing the state of the droplet changing near the disconnection position and the lateral flow trajectory opening and closing accordingly.

[0021] Figure 5 This is a view showing an example of a droplet observed at a frequency of 100 kHz in an environment where the room temperature decreased by 3.7 °C in 1 hour.

[0022] Figure 6 This is a view showing an example of a droplet observed at a frequency of 100 kHz with the room temperature variation limited to 23.4 ± 0.3 °C over 1 hour.

[0023] Figure 7 This is a diagram illustrating an example of a droplet becoming unstable at a specific frequency.

[0024] Figure 8 This is a flowchart illustrating an example of control of a particle separation device according to the present technology.

[0025] Figure 9 This is a diagram illustrating the state of a control example of a particle separation device according to the present technology.

[0026] Figure 10 This is a diagram showing the variation of the break point (BOP) length depending on the liquid temperature of the sheath fluid. Detailed Implementation

[0027] In the following description, preferred embodiments for carrying out this technology will be described with reference to the accompanying drawings.

[0028] The embodiments described below illustrate examples of representative implementations of the present technology, and the scope of the present technology is not limited thereto. Note that the descriptions will proceed in the following order.

[0029] 1. Overview of this technology

[0030] 2. First Embodiment (Particle Separation Device 1)

[0031] (1)Flow path P

[0032] (2) Vibration unit 11

[0033] (3) Imaging unit 12

[0034] (4) Liquid temperature control unit 13

[0035] (5) Frequency control unit 14

[0036] (6) Determine Unit 15

[0037] (7) Charging unit 16a, deflection plate 16b, recycling container 16c

[0038] (8) Disconnect control unit 17

[0039] (9) Detection Unit 18

[0040] (10) Analysis Unit 19

[0041] (11) Storage unit 20

[0042] (12) Display unit 21

[0043] (13) User Interface 22

[0044] (14) Other

[0045] 3. Examples of control flows

[0046] (1) Measurement parameter setting process (steps S101 to S103)

[0047] (2) BOP measurement process (steps S104 to S110)

[0048] (3) BOP data analysis process (steps S111 to S120)

[0049] (4) Effect

[0050] 4. Second Implementation Method (Particle Separation Method)

[0051] 1. Overview of this technology

[0052] This technology maintains a constant sideflow trajectory that transports particles to a recovery container in a device. The device illuminates the particles with light aligned in the flow path, detects the light emitted from each particle, and determines whether the droplet containing the particle is positively (+) or negatively (-), or uncharged. Based on the detection signal or whether the droplet is uncharged, the droplet is divided into corresponding droplet trajectories by a deflection plate, and the target particles are collected.

[0053] To maintain a constant lateral flow trajectory, precise droplet control with minimal temporal variation and fluctuation is required. Here, the droplets become charged in the aforementioned device at the moment they separate from the liquid column. Therefore, it is crucial to adjust the timing of droplet separation (also known as "disconnection") and the charging pulse. If disconnection is not performed within the pulse width of the charging signal, sufficient charge cannot be applied to the droplets, the deflection angle decreases proportionally to the amount of charge, and the lateral flow closes inward.

[0054] The charging pulse typically has a time width T equal to one droplet period, and therefore the timing is first adjusted so that the break-off time of the target particle falls within the charging pulse width T. However, in the case of a real charging pulse with a rise time (Tr) and fall time (Tf) of the charging signal, the effective pulse width (Te) at the maximum voltage (Vtop) is obtained by subtracting them from T: Te = T - (Tf + Tr). For example, when the frequency of the driving voltage is 100 kHz, one droplet period T is 10 μsec, and when both Tr and Tf are 3 μsec, Te is a value halved to 4 μsec. In a simplified manner, this Te value is a margin that allows for time variation during break-off. Figure 3 This illustrates the relationship between a droplet cycle and the correct timing of the charging signal.

[0055] Variations in the droplet's break point (also known as the "break point" or "BOP") can be observed in detail by illuminating the droplet with a light source that flashes in sync with the drive signal of the vibrating element and acquiring an image of the droplet using an imaging element. For example, Figure 4 The diagram illustrates the changing state of a droplet near its point of impact (BOP) and the corresponding opening and closing of the lateral flow trajectory when the frequency is set to 100 kHz and the droplet remains stationary for 2000 seconds. Figure 4 In this process, the phase of the charging pulse is adjusted to the droplet, causing the sideflow to open at its maximum angle at the start of observation. Figure 4 As can be seen, the disconnection timing advances with time, and specifically, the length and position of the auxiliary droplets located between the main droplets change. Then, after 2000 seconds, the lateral flow returns to its maximum angle because the advance in disconnection time essentially corresponds to the amount of one droplet cycle. However, the lower droplet, which initially needs charging, does not deflect; instead, the upper droplet, which is offset by a position, deflects.

[0056] To maintain a constant sideflow trajectory throughout the entire operation time of separating droplets containing the target particles (also known as "sorting"), it is necessary to maintain a level of change that is virtually undetectable in the aforementioned droplet observation images. Several droplet stabilization methods have been proposed in the past as a measure of this. Here, two representative methods will be described.

[0057] (i) Perform stabilization control on the droplet by referring to the droplet observation image.

[0058] (ii) Maintain a constant sheath fluid temperature.

[0059] Each method will be described in detail below.

[0060] In method (i) above, during the sorting process, the side-flow illumination is not always as expected because the device configuration blocks the recycling container or because the sorting frequency is very low. Figure 4 The lateral flow trajectory is clearly observed. Therefore, this method applies stable feedback control to the droplet using a droplet observation image. Specifically, using the droplet observation image at the start of the measurement as a reference, the droplet is adjusted by continuously applying feedback to the driving voltage of the vibrating element so that it does not change in the droplet observation image. Several suggestions have been made regarding the focal point of the droplet observation image, and for example, in Patent Document 1 mentioned above, the driving voltage of the vibrating element is controlled such that the distance between the end of the droplet just before it breaks apart and the end of an adjoining droplet at a previous position in the droplet observation image is constant. Furthermore, in Patent Document 2, the driving voltage of the vibrating element is controlled by focusing on the length of the first adjoining droplet.

[0061] Method (ii) above is based on the idea that the most influential droplet variation factor is ambient temperature change, aside from irregularities such as contamination by foreign objects or bubbles, and specifically, the sheath fluid temperature directly affects droplet variation. When the sheath fluid temperature changes due to variations in ambient temperature, heat generated by the device itself, etc., it imparts two different effects on the droplets. Here, Figure 5 An example of observing droplets at a frequency of 100 kHz in an environment where room temperature decreased by 3.7 °C in 1 hour is shown, and each effect will be described in detail.

[0062] The first is the change in flow velocity. The viscosity of the liquid changes with the temperature of the sheath fluid, causing an increase or decrease in pressure loss in the flow path and resulting in a variation in the velocity of the jet ejected from the orifice. For example, as a result of experiments conducted by the inventors of this application, when the temperature of the sheath fluid decreased by 4°C, the liquid viscosity increased by 8%, and the flow velocity of the 30 m / s jet decreased by approximately 2%. At this time, although the BOP is slightly altered to the point of shortening by 20 to 30 μm, the disconnection time remains unchanged, preventing timing deviation from the charging signal and not affecting the sideflow trajectory.

[0063] The second is the change in disconnection timing. The disconnection time can be rapidly changed due to variations in the temperature of the sheath fluid. Figure 5In this study, when the room temperature drops by approximately 2°C within the first 30 minutes, the breakup time is advanced by approximately two wavelengths (20 μsec). This change directly affects the sideflow trajectory and is therefore a major problem. Unlike the first phenomenon described above, which can be reproduced based on the physical properties of the sheath fluid, this phenomenon is considered to be attributed to the vibrational characteristics of the entire system. Therefore, this phenomenon exhibits random behavior dependent on the device state and droplet formation conditions, and is very difficult to handle. The problem in this technology is the BOP variation caused by the second phenomenon.

[0064] here, Figure 6 An example of droplet observation at a frequency of 100 kHz is shown, where the room temperature variation over 1 hour is limited to 23.4 ± 0.3 °C. (Compared to...) Figure 5 In comparison, it can be seen that changes in flow velocity and disconnection timing are greatly limited. However, maintaining a constant sideflow trajectory requires more precise droplet control.

[0065] As mentioned above, since the temperature variation of the liquid in the sheath fluid has a large impact, Patent Document 3 proposes a technique for controlling the liquid temperature of the sheath fluid in a sheath fluid introduction unit that introduces the sheath fluid into the flow path to prevent droplet instability caused by changes in the flow rate of the sheath fluid due to changes in ambient temperature, although the main purpose is to stabilize the flow rate during measurement.

[0066] As droplet stabilization methods, both methods (i) and (ii) above are correct and effective. However, there are cases where droplets become significantly unstable to the point that even these measures cannot control them, and this situation must be avoided in advance. Prior to methods (i) and (ii) above, this technique proposes a method for finding droplet stability conditions that are originally highly tolerant of changes in ambient temperature.

[0067] Here, in Figure 7 An example of droplet instability at a specific frequency is shown. Figure 7 In this study, under a liquid supply pressure of 600 kPa, the frequency characteristics of the BOP were measured three times in 1 kHz steps by continuously forming droplets within a range of 100 ± 20 kHz while the droplet frequency was varied. Note that the driving voltage of the vibrating element was fixed, and no feedback control was applied. The test chamber temperature was 25.6 °C in the first measurement, 26.5 °C in the second measurement, and 26.8 °C in the third measurement. Figure 7 An assessment of droplet stability is shown when the ambient temperature changes by 1.2°C over a period of approximately two hours. Additionally, in Figure 7 In the graph shown, the X-axis represents the droplet frequency, and the Y-axis represents the BOP (position coordinate: the larger the value, the shorter the actual BOP length).

[0068] like Figure 7 As shown, the following frequencies exist: frequencies where the BOP remains almost unchanged for a temperature change of approximately 1.2°C over two hours; and another frequency where the BOP changes significantly. The former includes frequencies such as 85 to 92 kHz and 95 to 101 kHz, which can be considered frequencies that are stable relative to temperature changes. On the other hand, the latter includes frequencies such as 80 to 82 kHz, 93 to 94 kHz, and 105 to 106 kHz. Specifically, at 105 to 106 kHz, the BOP changes significantly, by approximately 5 mm, which can be considered an anomalous behavior corresponding to a one-position change in the amplitude of the vibrating element. Therefore, in cases where such a level of BOP variation occurs, conventional droplet stabilization methods may not be able to handle the situation. Specifically, in method (i) above, when the BOP variation is too large, the BOP variation may exceed the adjustment range of the driving voltage and become uncontrollable. Furthermore, in method (ii) above, it is practically difficult to completely suppress the temperature variation of the liquid in the sheath fluid to zero.

[0069] For the reasons mentioned above, it can be said that it is desirable to control the frequency of time-related changes in ambient temperature, etc., in order to control the sideflow trajectory with greater accuracy and stabilize the droplets.

[0070] 2. First Embodiment (Particle Separation Device 1)

[0071] Figure 1 This is a schematic diagram illustrating a configuration example of a first embodiment of the particle separation apparatus 1 according to the present technology. Furthermore, Figure 2 This is a schematic diagram illustrating another configuration example of a particle separation device according to the present technology, representing a first embodiment.

[0072] Figure 1 and Figure 2 The particle separation device 1 shown includes at least a vibration unit 11, an imaging unit 12, a liquid temperature control unit 13, and a frequency control unit 14. Furthermore, the particle separation device 1 may, as needed, include a flow path P, a determination unit 15, a charging unit 16a, a deflection plate 16b, a recovery container 16c, a disconnection control unit 17, a detection unit 18, an analysis unit 19, a storage unit 20, a display unit 21, and a user interface 22, etc.

[0073] (1)Flow path P

[0074] A fluid containing at least sheath fluid flows through flow path P. Furthermore, a sample solution containing particles and a sheath fluid flowing to surround the sample solution can flow as needed, and in this case, flow path P can be configured to form a flow in which the particles are arranged in a substantially linear manner. Flow path P can be pre-installed in the particle separation device 1, but commercially available flow paths, disposable chips with flow paths, etc., can also be installed.

[0075] The form of the flow path P is not particularly restricted and can be designed freely as appropriate. For example, the flow path is not limited to... Figure 1 The flow paths shown are formed in substrates such as two-dimensional or three-dimensional plastics or glass, and can also be used in... Figure 2 The flow path used in a conventional flow cytometer is shown in the figure.

[0076] The flow path width, flow path depth, and flow path cross-sectional shape of the flow path P are not particularly restricted and can be designed freely. For example, a microflow path with a channel width of 1 mm or less can also be used in the particle separation device 1.

[0077] In this technology, specifically, microflow paths with a flow path width of about 10 μm and less than 1 mm can be appropriately used.

[0078] (2) Vibration unit 11

[0079] The vibration unit 11 applies vibration to the fluid containing sheath fluid by supplying a driving voltage based on at least two or more frequencies. Therefore, the fluid can continuously form droplets to generate a fluid flow (droplet flow). The multiple frequencies can be user-specified frequency domains. This embodiment can be applied to chip systems and flow cell systems, including, in chip systems, such as... Figure 1 The entire flow path system of the nozzle shown is integrated and replaceable, in a flow pool system, such as... Figure 2 The flow path system shown is fixed and only the tip nozzle is replaceable.

[0080] Vibration is applied by, for example, a vibrating element 111. The vibrating element 111 is not particularly limited and can be any conventionally known vibrating element, specifically a piezoelectric element, etc. When the chip T is used as the flow path P, the vibrating element 111 is preferably disposed near the aperture O of the chip T.

[0081] The driving voltage is supplied to the vibration unit 11 via, for example, a voltage supply unit. For example, the driving voltage is provided according to a sine wave to form a stable droplet D, and the driving voltage is controlled by two factors: frequency (clock value) and amplitude (driving value).

[0082] exist Figure 1In the illustrated chip system, the sheath fluid inlet and sheath fluid flow paths P12a and P12b, the sample fluid inlet and sample fluid flow path P11, the confluence of these flow paths and the main flow path P13 irradiated by light, and the orifice O (outlet nozzle) are integrated and replaceable. The sample fluid flow path P11 is linearly arranged at the center, and the sheath fluid flow paths P12a and P12b branch to the left and right from the inlet to surround the sample fluid flow path P11, and the three flow paths eventually converge to become the main flow path P13. As a result, laminar flow is formed, such that the sample fluid is sandwiched in the middle by the sheath fluid and travels to the linear flow path section for light irradiation detection. In this case, when vibration is applied to a portion of the substrate surface forming the chip T by the vibration element 111, the droplet D is formed by the liquid column L ejected from the orifice O. Alternatively, the sheath fluid can be vibrated directly before the inlet of the chip T.

[0083] In such Figure 2 In the flow cell system shown, the sheath fluid and sample solution are injected into a conical container. The conical container is mounted with its apex pointing vertically downwards, and a tube for introducing the sheath fluid is connected to the upper surface. The upper surface of the conical container is open, and the vibrating element 111 is attached in a sealed state with an O-ring. The sample solution is injected vertically from above the container. The conical container narrows at its lowest point, and its tip connects to the cuvette section, which forms the main flow path (linear flow path) P13. When laminar flow is formed such that the sheath fluid surrounds the sample solution in the conical container and the sample solution flows as a laminar stream into the cuvette section, detection by light irradiation is performed in the main flow path P13. A removable outlet nozzle is installed at the end of the main flow path P13, and the connection portion has an inclined shape that continuously narrows from the outlet of the cuvette to the outlet nozzle. The sheath fluid and sample solution vibrate slightly in the forward and backward direction relative to the flow from the vibrating element 111 mounted directly above the conical container. Then, the liquid column L ejected from the outlet nozzle travels vertically downwards, widening into a crack at the same frequency as the vibration generated by the vibrating element 111, and forming a droplet at the break point at the nozzle outlet.

[0084] (3) Imaging unit 12

[0085] Imaging unit 12 acquires images of the fluid and droplets at locations where the fluid is formed into droplets through vibration. Specifically, at the break point where the sheath fluid discharged from orifice O forms droplets, images of the fluid and droplets before droplet formation are captured.

[0086] Specific examples of the imaging unit 12 include a droplet camera 121, such as a charge-coupled device (CCD) camera or a complementary metal-oxide-semiconductor (CMOS) sensor. The droplet camera 121 can be positioned to image the droplet D between the aperture O and the deflection plate 16b, described later. Furthermore, the droplet camera 121 can adjust the focus of the captured image of the droplet D. Examples of light sources illuminating the imaging area in the droplet camera 121 include a flash lamp 122, etc. Note that the imaging unit 12 can also acquire a phase photograph at a certain time, and can acquire the photograph continuously within a specific period. The term "specific period" mentioned herein is not particularly limited and can be one period or multiple periods. In the case of multiple periods, each period can be temporally continuous or discontinuous.

[0087] The image captured by the droplet camera 121 is displayed on the display unit 21, which will be described later, and the user can use the image to confirm the formation state of the droplets D (e.g., the size, shape, spacing, etc. of the droplets D).

[0088] The flash lamp 122 can be controlled by the disconnect control unit 17, which will be described later. The flash lamp 122 includes, for example, a light-emitting diode (LED) for imaging the droplet D and a laser (e.g., a red laser source, etc.) for imaging the particle, and can be switched by the disconnect control unit 17 according to the purpose of imaging, etc. The specific structure of the flash lamp 122 is not particularly limited, and one or more conventionally known circuits or components can be appropriately and freely selected and used.

[0089] (4) Liquid temperature control unit 13

[0090] The liquid temperature control unit 13 controls the liquid temperature of the sheath fluid. The liquid temperature control unit 13 may include, for example, a temperature sensor that measures the liquid temperature of the sheath fluid and a temperature control unit. The temperature sensor and the temperature control unit are connected by a circuit. The installation location of the temperature sensor is not particularly restricted and it can be in direct contact with the sheath fluid. However, to prevent contamination, the temperature sensor is preferably installed at a location where the liquid temperature of the sheath fluid can be measured indirectly. Specifically, at the end of the tube through which the sheath fluid flows, immediately before the inlet of the flow cell or chip T, a material with high thermal conductivity (e.g., a metal tube) is inserted, and the temperature sensor is mounted thereon. In this case, by further covering the sensor surface with a thermally insulating material to prevent the temperature sensor from being exposed to outside air, the liquid temperature of the sheath fluid can be measured indirectly with high accuracy. Furthermore, if there is a difference between the temperature detected by the temperature sensor and the target temperature to be achieved, the liquid temperature control unit 13 can take this difference into account to correct the target temperature of the temperature checking unit, which will be described later.

[0091] Furthermore, when the sample liquid containing particles and the sheath fluid flow through flow path P, the temperature sensor is preferably located near the confluence of the sample liquid and the sheath fluid. This allows the liquid temperature of the sheath fluid to be maintained even after sorting has begun, and suppresses droplet instability caused by temperature fluctuations in the sheath fluid. Additionally, this is because when located too far from the flow path, the temperature sensor may be affected by ambient temperature during that time. Alternatively, in this case, the temperature sensor can be a different sensor than the one located in the liquid temperature control unit 13.

[0092] The temperature control unit may include, for example, a temperature control element, a metal component disposed in contact with the temperature control element, and a temperature sensing unit for checking the temperature of the metal component. The temperature control element may be, for example, a semiconductor cooling element that generates a temperature difference by applying a direct current; specific examples include Peltier elements. The metal component is not particularly limited and may be made of, for example, copper or brass. The metal component may be disposed in contact with, for example, a tubular component supplying sheath fluid. The temperature sensing unit is mounted on the metal component and detects or controls the temperature of the metal component to achieve a target temperature. Furthermore, the temperature control unit may also include a heat sink and a fan.

[0093] (5) Frequency control unit 14

[0094] Although details will be described later, the frequency control unit 14 acquires data on the state of droplet D at each frequency for each liquid temperature of the sheath fluid from the image captured by the imaging unit 12, and controls the frequency of the drive voltage based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

[0095] Examples of data regarding the state of droplet D include the size of droplet D, the type of adjoining droplets of droplet D, the state of the liquid column L, adjoining droplets and main droplet, the adhesion state between droplet D and liquid column L, the distance between droplet D and liquid column L, the break point of droplet D (including the concept of "BOP length"), etc., and preferably the break point of droplet D.

[0096] (6) Determine Unit 15

[0097] As will be described in detail later, when the difference between the break points of the droplets is equal to or greater than a threshold, the determining unit 15 determines the corresponding frequency as an "unstable frequency". Furthermore, when the difference between the break points of the droplets is less than the threshold, the determining unit 15 determines the corresponding frequency as a "stable frequency". Additionally, the frequency with the smallest difference between the break points of the droplets can be determined as the "most stable frequency".

[0098] (7) Charging unit 16a, deflection plate 16b, recycling container 16c

[0099] Charging unit 16a charges droplet D containing target particles discharged from orifice O. In this technology, droplet D containing target particles is formed by supplying a driving voltage based on a frequency other than the frequency determined as an "unstable frequency" by the determining unit 15 described above. This allows for the early elimination of unstable frequencies, enabling sorting to be performed when the droplets are in a stable state.

[0100] In this technology, "particles" can broadly include biological particles (such as cells, microorganisms, and ribosomes) or synthetic particles (such as latex particles, gel particles, and industrial particles). Furthermore, in this technology, particles can be contained in fluids such as liquid samples.

[0101] Biologically relevant particles can include chromosomes, ribosomes, mitochondria, organelles, etc., that form various cells. Cells can include animal cells (e.g., blood cells) and plant cells. Microorganisms can include bacteria (e.g., Escherichia coli), viruses (e.g., tobacco mosaic virus), fungi (e.g., yeast), etc. Furthermore, biologically relevant particles can also include, for example, biologically relevant polymers such as nucleic acids, proteins, and their complexes.

[0102] Industrial particles can be, for example, organic or inorganic polymer materials, metals, etc. Organic polymer materials can include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials can include glass, silica, magnetic materials, etc. Metals can include gold colloids, aluminum, etc. Typically, these particles are spherical in shape, but in this technology they can be non-spherical, and their size, mass, etc., are not particularly limited.

[0103] In this technology, biologically relevant particles are preferred among these particles.

[0104] In this technique, particles can be labeled with one, two, or more dyes (such as fluorescent dyes). Available fluorescent dyes include, for example, Cascade Blue, Pacific Blue, FITC, phycoerythrin (PE), propidium iodide (PI), Texas Red (TR), piperidine chlorophyll protein (PerCP), allophycocyanin (APC), diphenylamine with tetrazole (4',6-Diamidine-2-phenylindole, DAPI), Cy3, Cy5, Cy7, Brilliant Violet (BV421), etc.

[0105] As a method for separating droplets D containing target particles, for example, based on a detection signal obtained from light irradiation from light source 181 described later, droplets D generated by vibration from the aforementioned vibration unit 11 are charged positively or negatively as needed by charging unit 16a, and the direction of advance of droplet D2 is controlled by the presence or absence of electricity between deflection plates 16b and the magnitude of the electricity, and is guided to a predetermined recovery container 16c.

[0106] The charging unit 16a may include a charging electrode, a voltage source (applying a desired voltage to the electrode), etc. The charging electrode may be configured to make electrical contact with the sheath fluid flowing through the flow path P and may be inserted into the charging electrode inlet of the chip T, for example, as... Figure 1 As shown.

[0107] The deflecting plates 16b deflect the direction of travel of each droplet D in the liquid flow by the electric current acting between the deflecting plates and the charge applied to the droplets D, and guide the droplets D to a predetermined recovery container 16c, etc., and are arranged to face each other across the liquid flow S. Figure 1 In this diagram, the orientation of the deflection plate 16b is indicated by the X-axis direction. The deflection plate 16b is not particularly limited and can use conventionally known electrodes, etc. Different positive or negative voltages are applied to the deflection plate 16b, and when charged droplets D pass through the electric field they form, an electric current (Coulomb force) is generated, and each droplet D is attracted toward either deflection plate 16b.

[0108] Multiple recycling containers 16c can be positioned along the facing direction of the deflection plate 16b. Figure 1 The X-axis direction (in the image) is arranged in a basically linear manner. There are no particular limitations on the recycling containers 16c; examples include plastic tubes, glass tubes, etc. The number of recycling containers 16c is also not particularly limited. Figure 1 and Figure 2 Examples of three recycling containers installed are shown in each example. Note that recycling container 16c can be installed in a replaceable manner within a recycling container housing (not shown). Specifically, for example, the recycling container housing can be positioned on a Z-axis stage (not shown), which is configured to be aligned with the discharge direction of the droplets D exiting from orifice O ( Figure 1 The Y-axis direction in the image) and the facing direction of the polarizer 16b ( Figure 1 The direction orthogonal to the X-axis (in the middle) Figure 1 It can move along the Z-axis.

[0109] (8) Disconnect control unit 17

[0110] The disconnection control unit 17 controls the disconnection of the droplet D containing the target particles based on an image of the state of the droplet D containing the particles acquired by the imaging unit 12. Specifically, the driving voltage of the vibrating element 111 is adjusted based on the disconnection timing of the droplet D containing the particles, specified by multiple droplet observation images captured by the imaging unit 12, thereby controlling the adhesion state between the droplet D and the liquid column L and / or the distance between the droplet D and the liquid column L, and keeping the disconnection position of the droplet D constant. Therefore, by continuously applying feedback to the driving voltage to adjust the droplet, droplet instability after the start of sorting can be suppressed.

[0111] (9) Detection Unit 18

[0112] The detection unit 18 detects the measurement target light generated from particles flowing through the flow path P by irradiating them with light emitted from the light source 181. Specifically, by irradiating the particles with light, the detection unit 18 detects the measurement target light generated from the particles, which is aligned in a generally linear manner at the center of the three-dimensional laminar flow in the main flow path P13 while being delivered.

[0113] Light source 181 may include multiple light sources, and in this case, light from multiple light sources can be used to illuminate particles in the fluid flowing in the flow path P. Furthermore, each of the multiple light sources may emit light with the same wavelength or may emit light with different wavelengths.

[0114] There are no particular restrictions on the type of light emitted from light source 181, but in order to reliably generate light from particles, it is desirable to have light with constant direction, wavelength, and intensity. Specific examples include laser beams, LEDs, etc.

[0115] Examples of lasers include semiconductor lasers, argon-ion (Ar) lasers, helium-neon (He-Ne) lasers, dye lasers, krypton (Cr) lasers, solid-state lasers combining semiconductor lasers and wavelength-converting optics, and two or more of them can be used in combination.

[0116] The detection unit 18 includes at least one photodetector for detecting the target light being measured. Examples of the target light being measured include fluorescence, scattered light (e.g., forward scattered light, backscattered light, side scattered light, Rayleigh scattering, Mie scattering, etc.). For example, each photodetector includes one or more light-receiving elements and has an array of light-receiving elements. Each photodetector may include one or more photodiodes, such as photomultiplier tubes (PMTs) and / or avalanche photodiodes (APDs), and a multi-pixel photon counter (MPPC) as a light-receiving element. The photodetector may include, for example, an array of PMTs arranged in a one-dimensional direction. Furthermore, the detection unit 18 may include an imaging element such as a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS). The target light being measured is converted into an electrical signal by the photodetector, and the electrical signal is output to the determination unit 15 and the disconnection control unit 17, the analysis unit 19 described later, etc., and used for the determination of the characteristics of each particle.

[0117] In addition to the light source 181, the detection unit 18 may include a light-guiding optical system for guiding multiple beams to predetermined positions, a detection optical system for directing light with predetermined detection wavelengths to corresponding photodetectors, etc. For example, the light-guiding optical system may include optical components (such as beam splitter groups, mirror groups, and optical fibers). Furthermore, the light-guiding optical system may include lens groups for collecting multiplexed excitation light, and may include, for example, an objective lens. The detection optical system may include a beam-splitting unit (such as a prism or diffraction grating) or a wavelength-separating unit (such as a dichroic mirror or optical filter).

[0118] It should be noted that the detection unit 18 can detect the characteristics of the particles magnetically or electrically. In this case, for example, microelectrodes are arranged facing each other in the main path P13 of the chip T, and resistance, capacitance, inductance, impedance, changes in the electric field or magnetization between the electrodes, changes in the magnetic field, changes in flux density, etc., can be detected.

[0119] (10) Analysis Unit 19

[0120] The analysis unit 19 is connected to the imaging unit 12, the detection unit 18, etc., and performs analysis based on the image acquired by the imaging unit 12 and the electrical signal detected by the detection unit 18.

[0121] Specifically, the analysis unit 19 analyzes or calculates data on the state of the droplet D from the image acquired by the imaging unit 12. Furthermore, based on the data regarding the state of the droplet D, the change in BOP ΔBOP (difference in break position), etc., can be obtained.

[0122] Furthermore, the analysis unit 19 can correct the detection values ​​detected by the detection unit 18 and calculate the characteristic quantities of each particle. For example, it can calculate characteristic quantities such as particle size, shape, and internal structure based on the received fluorescence or scattered light detection values. Additionally, a separation control signal can be generated by performing separation determination based on the calculated characteristic quantities and separation conditions received from the user interface 22, which will be described later. By controlling the charging unit 16a based on the separation control signal, specific types of particles can be separated and collected.

[0123] The analysis unit 19 may be housed within a casing that includes the detection unit 18, or it may be located outside the casing. Furthermore, the particle separation apparatus 1 according to this embodiment is not essential, and external analysis devices may also be used. Additionally, the analysis unit 19 may be connected to each unit of the particle separation apparatus 1 via a network.

[0124] (11) Storage unit 20

[0125] Storage unit 20 stores all items related to analysis and separation, such as the detection values ​​detected by detection unit 18, the feature quantities calculated by analysis unit 19, the generated separation control signals, and the separation conditions input by user interface 22.

[0126] The storage unit 20 may be included in the housing on which the detection unit 18 is installed, or it may be located outside the housing. Furthermore, the storage unit 20 is not essential to the particle separation apparatus 1 according to this embodiment, and external storage devices (e.g., hard disks) may also be used. Additionally, the storage unit 20 may be connected to each unit of the particle separation apparatus 1 via a network.

[0127] (12) Display unit 21

[0128] Display unit 21 can display all items related to analysis and separation, and for example, can display the characteristic quantities of each particle calculated by analysis unit 19 as histograms, etc. Furthermore, it can also display images captured by imaging unit 12, etc.

[0129] The display unit 21 is not necessary in the particle separation apparatus 1 according to this embodiment, and an external display device (e.g., a monitor, printer, personal digital assistant, etc.) can also be used. Furthermore, the display unit 21 can be connected to each unit of the particle separation apparatus 1 via a network.

[0130] (13) User Interface 22

[0131] User interface 22 is a component operated by the user. The user can input various data through user interface 22 and access each unit of particle separation device 1 to control each unit. For example, the user can set the region of interest, such as the histogram displayed on display unit 21, and determine separation conditions, etc., via user interface 22.

[0132] In the particle separation apparatus 1 according to this embodiment, the user interface 22 is not necessary, and external operating devices (e.g., mouse, keyboard, personal digital assistant, etc.) can also be used. Furthermore, the user interface 22 can be connected to each unit of the particle separation apparatus 1 via a network.

[0133] (14) Other

[0134] It should be noted that the functions performed in each unit of the particle separation apparatus 1 according to the present technology can also be stored as programs in a general-purpose computer, a control unit including a CPU, and hardware resources including recording media such as non-volatile memory (e.g., USB memory), HDD, and CD, and the functions can be executed. Furthermore, the functions can be implemented via a server computer or the cloud connected via a network.

[0135] 3. Examples of control flows

[0136] Next, the description will be based on Figure 1 and Figure 2 An example of control performed by the particle separation device 1 in the embodiment shown. Figure 8 This is a flowchart illustrating a control example. Furthermore, Figure 9 This is a diagram showing the state of a control example, with the downward arrows indicating unstable frequencies. It should be noted that the execution of the operations described later can be controlled by a control unit (not shown) or similar device controlling the particle separation apparatus 1.

[0137] The control flow example described below coexists with the conventional droplet stabilization methods described above, and can, for example, be inserted in the pre-stage of the conventional methods. In this case, the burden in the conventional droplet stabilization methods can be reduced, and the effects of these methods can be further demonstrated.

[0138] (1) Measurement parameter setting process (steps S101 to S103)

[0139] First, parameters related to droplet formation, other than the frequency (mainly the sheath fluid supply pressure and driving voltage), are determined (step S101). At this time, the sheath fluid supply pressure is preferably the same as the sheath fluid supply pressure during sorting. On the other hand, the driving voltage is adjusted again according to the frequency selected before starting sorting; therefore, the driving voltage does not need to be the same at this stage.

[0140] Next, to measure the disconnection position by changing the sheath fluid temperature in several stages, multiple fluid temperature settings [T1, T2, ..., Tx] (x ≥ 2) are entered (step S102). At this point, if the number of fluid temperature settings increases excessively, time will be required for the temperature to stabilize when the fluid temperature settings are changed, increasing the required time. Therefore, unless a detailed investigation of BOP variation is particularly needed, for example, three points—maximum, center, and minimum—can be entered within an assumed temperature variation (e.g., ±3°C) relative to room temperature at the start of the measurement or within a predetermined range of settings. Figure 9 In the example shown, the liquid temperature of the sheath fluid was set to three stages: 24°C, 28°C, and 32°C.

[0141] Next, the measurement start frequency (fb), measurement end frequency (fe), and measurement frequency step size (Δf) are set (step S103). Typically, when determining the nozzle diameter and sheath fluid supply pressure, there exists an optimal frequency fs based on physical properties (surface tension), therefore, it is preferable to study a range of ±10% to 20% around this fs. Furthermore, in step S103, similar to step S102, if the frequency range is too wide or the frequency step size is too small, the number of BOP measurement points increases, thus requiring excessive time. Therefore, depending on the speed of BOP measurement, for example, the number of BOP measurement points can be set to approximately 10 per sheath fluid temperature point.

[0142] (2) BOP measurement process (steps S104 to S110)

[0143] Using the parameters (sheath fluid supply pressure, driving voltage, and measurement start frequency fb) input during the above measurement parameter setting process, the vibration unit 11 applies vibration to the fluid containing only sheath fluid to form droplets D (step S104). Note that during this BOP measurement process, only sheath fluid may be allowed to flow through the flow path P, and it is not necessary to allow the sample fluid containing particles to flow through the flow path P.

[0144] Next, the liquid temperature control unit 13 sets the liquid temperature of the sheath fluid to temperature T1 and waits for the liquid temperature to stabilize (step S105). When the liquid temperature stabilizes, data on the disconnection position is acquired for each specified frequency [fb, fb+Δf, fb+2Δf,,,fe] (step S106). The data can be acquired by analyzing the image captured by the imaging unit 12 in the analysis unit 19. Furthermore, if the BOP can be tracked by moving the position of the droplet camera 121, the BOP can be calculated by assigning the Z position of the camera 121.

[0145] Next, the liquid temperature control unit 13 sets the liquid temperature of the sheath fluid to temperature T2 and waits for the liquid temperature to stabilize again (step S107). When the liquid temperature stabilizes, the disconnection position data is acquired again for each specified frequency [fb,fb+Δf,fb+2Δf,,,fe] (step S108).

[0146] Subsequently, the liquid temperature of the sheath fluid is set to temperature Tn+1 (steps S109 to S110), and step S108 is repeated until the liquid temperature of the sheath fluid reaches temperature Tx.

[0147] (3) BOP data analysis process (steps S111 to S120)

[0148] Analysis unit 19 analyzes the disconnection position data obtained during the BOP measurement process described above and obtains the BOP variation (ΔBOP) caused by the change in liquid temperature [T1, T2, ..., Tx] at each frequency (the difference in disconnection position) (step S111). Next, an allowable BOP variation (Bth) is set (step S112). For example, when droplet stabilization control is performed by feedback to the drive voltage, there is a risk of the voltage exceeding the output voltage range when a large voltage variation range of approximately ±50% relative to the initial value of the drive voltage is required. Therefore, considering the BOP variation for one droplet before and after, Bth is set, which is a range within which the sideflow trajectory can be easily stabilized by feedback control. Specifically, for example, since the standard droplet spacing is known to be approximately nozzle diameter × 4.5, Bth can be arbitrarily determined from its relationship with the nozzle diameter used for measurement. Figure 9 In the example shown, Bth = 0.6 mm is set.

[0149] Subsequently, the determination unit 15 compares ΔBOP with Bth for each frequency (step S113). When ΔBOP ≥ Bth (Yes in step S114), the determination unit 15 determines that the corresponding frequency is an "unstable frequency," and based on the determination result, the frequency control unit 14 excludes the unstable frequency from the frequency options that can be used as a drive voltage (step S115). On the other hand, when ΔBOP < Bth (No in step S114), the determination unit 15 determines that the corresponding frequency is a "stable frequency," and based on the determination result, the frequency control unit 14 can include the stable frequency from the frequency options that can be used as a drive voltage (step S118). That is, the frequency control unit 14 can use any frequency other than the unstable frequency excluded in step S115 as a stable frequency. This allows the sideflow trajectory to stabilize. Figure 9 In the example shown, the frequencies indicated by the down arrows are those identified as "unstable frequencies".

[0150] In addition, examples of other criteria for selecting the frequency include the event rate related to sorting performance, the probability of duplex formation (a state containing multiple particles in a droplet), etc. Furthermore, regarding droplets, there are indicators such as the ability to obtain a suitable disconnection position under a suitable driving voltage, or the more stable FAST-associated states that may result in sideflow trajectories. Therefore, users can comprehensively determine these factors and thus determine the final frequency.

[0151] Alternatively, if the device has an existing frequency selection algorithm, unstable frequencies can be excluded in step S115, and then processing can be performed continuously. An example of the algorithm is the method described in Japanese Patent Application Publication No. 2020-76786A. In this method, the guideline for frequency selection is that the BOP length is as short as possible, and the adjoint is collected in the main droplet within an arbitrarily specified distance range. By using this algorithm in conjunction with this technology, the stability of the sideflow trajectory over time can be guaranteed.

[0152] Next, if the user wants to emphasize droplet stability, the most stable frequency is obtained according to the stable frequency option ("Yes" in step S119), and the frequency control unit 14 can adopt the frequency with the smallest ΔBOP as the most stable frequency (step S120). This is useful, for example, in situations where the reliability of the accuracy of the sheath fluid temperature control is low (e.g., where the ambient temperature variation may be large and exceed the processing capacity of the temperature control unit 13) or other situations. Specifically, practical devices are often used in the sealed environment of a safety cabinet, where the temperature inside the cabinet may continue to rise over time due to heat dissipation from the temperature control unit 13, etc. Therefore, adopting the most stable frequency is particularly effective in environments that take into account such liquid temperature variations.

[0153] Note that when ΔBOP is equally small at all frequencies, there are sometimes multiple most stable frequencies.

[0154] On the other hand, if the user wants to emphasize classification performance, an appropriate frequency can be adopted according to the option of each stable frequency that satisfies ΔBOP<Bth (step S116).

[0155] (4) Effect

[0156] Through the above processing, frequencies that are unstable with respect to ambient temperature over time can be detected in advance and excluded from the options of frequencies that can be used as driving voltages. Therefore, a constant sideflow trajectory can be maintained for a long time. Furthermore, by using a combination of conventional droplet stability control methods (e.g., feedback control of the driving voltage as described in Patent Document 1 above and temperature control of the sheath fluid as described in Patent Document 2 above), the burden on these methods can be reduced, providing stronger droplet stability, maintaining a constant sideflow trajectory for a long time, and stably guiding the particles to be sorted into the recovery container.

[0157] Furthermore, when necessary, the most stable frequency within a specified frequency range to changes in ambient temperature can be studied before classification. For example, even when the ambient temperature varies greatly, the processing capacity of the temperature control unit 13 cannot cope with the change, and a constant liquid temperature cannot be maintained, droplet stabilization can be achieved by selecting a frequency and combining it with feedback control of the drive voltage of the vibrating element.

[0158] Furthermore, if the device has an existing frequency selection algorithm, a more optimized frequency can be automatically determined by eliminating unstable frequencies before processing is performed.

[0159] In addition, depending on the setup conditions, the above series of control processes can be easily executed within the required approximately 20 minutes, enabling stable operation to be reliably achieved by selecting the optimal frequency before each execution sequence, even with different devices, changes in ambient temperature, or changes in device status.

[0160] 4. Second Implementation Method (Particle Separation Method)

[0161] The particle separation method according to this embodiment performs at least a vibration step, an imaging step, a liquid temperature control step, and a frequency control step. Additionally, a judgment step, a charging step, etc., may be performed as needed. It should be noted that the specific methods performed in each step are similar to those performed in each unit of the particle separation apparatus 1 according to the first embodiment described above, and therefore their description is omitted here.

[0162] In the particle separation method according to this embodiment, after the determination step, a charging step is performed to charge the droplets containing the target particles. In the charging step, droplets containing the target particles are formed by supplying a driving voltage based on a frequency other than an unstable frequency, causing vibrations. That is, by performing the determination step before starting the sorting process, errors in each device, variations in ambient temperature, and changes in device state can be addressed, and particles can be separated with higher accuracy.

[0163] However, maintaining the trend of frequency behavior while keeping the state of the device unchanged means that it is not always necessary to perform the determination step before each sequencing start. On the other hand, for example, when the orifice O of the chip T is removed due to cleaning or replacement, or when components of the flow cell are replaced, the determination step is preferably performed whenever possible. In particular, in the case of a chip system, it is recommended to perform the processing after loading a new chip T, because individual differences in the reproducibility of the chip T and clamping can be factors that alter the trend of frequency behavior.

[0164] [Example]

[0165] The present technology will be described in more detail below based on examples. Note that the examples described below illustrate representative implementations of the present technology, and the scope of the present technology is not limited by these examples.

[0166] <1> Test methods

[0167] Use such as Figure 2 The flow cell system shown searches for unstable and most stable frequencies.

[0168] With a nozzle diameter of 70 μm and a liquid supply pressure of 600 kPa, a jet velocity of 30.0 m / s was obtained. The piezoelectric drive voltage was fixed (amplitude ±8 nm), and the BOP length (Step_Δf = 1 kHz) was measured in the range of 100 ± 20 kHz.

[0169] The sheath fluid temperature was set in five stages (T1 = 23℃, T2 = 24℃, T3 = 25℃, T4 = 26℃, T5 = 27℃).

[0170] <2> Test Results

[0171] Figure 10 The results show the variation of BOP length with sheath fluid temperature at each frequency. Note that since the vertical axis of this graph is "BOP length", the lower side of the graph is larger than the lower side. Figure 9 It is shorter than the one in the middle.

[0172] Figure 10 The results shown indicate that there are large and small ΔBOP, with the frequency band having large BOP variation existing at approximately 95 to 100 kHz, while the frequencies with almost no BOP variation exist at approximately 80 kHz and 112 kHz.

[0173] <3> Detection of unstable frequencies

[0174] The threshold (Bth) used to determine the unstable frequency was set at approximately one droplet position before and after the unstable frequency, and here, Bth = 0.65 mm. As a result, the following frequencies satisfying ΔBOP ≥ Bth were detected as unstable frequencies.

[0175] 91,92kHz

[0176] ·96 to 100 kHz

[0177] Therefore, by selecting frequencies other than these unstable frequencies, the sideflow trajectory can be stabilized with high accuracy during the measurement period.

[0178] <4> Determine the most stable frequency

[0179] For all frequencies within the studied range of 100 ± 20 kHz, the frequency with the smallest ΔBOP was 111 kHz. This ΔBOP is 26 μm, and since ΔBOP tends to increase with increasing liquid temperature, it is considered to be a change approximately equal to the flow rate variation. Therefore, considering the measurement error of BOP, 80 kHz, 107 kHz, and 112 kHz, with ΔBOP < 100 μm, can also be considered to have similar stability and are candidates for selection as the most stable frequencies. Given that droplet stability relative to temperature is paramount during sorting, these most stable frequencies were used to determine the measurement frequency.

[0180] <5> other

[0181] Note that in this test example, the time required to measure the BOP length is 12 minutes for each temperature parameter stage. The settling time is four minutes for each 1°C increase in sheath temperature, and the automatic measurement time is eight minutes for every 41-point frequency measurement.

[0182] The number of frequency measurement points can be reduced and can be set, for example, ±12kHz (Δf = 2kHz). In this case, the number of measurement points is 13, approximately one-third of the original number, reducing the automatic measurement time to about 2.5 minutes. Furthermore, for example, when the temperature is set in three stages, the required time can be further reduced.

[0183] It should be noted that this technology can also be configured as follows.

[0184] [1] A particle separation device, comprising:

[0185] The vibration unit applies vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies;

[0186] The imaging unit acquires images of the fluid and droplets at the location where the fluid is formed into droplets by vibration;

[0187] A liquid temperature control unit controls the temperature of the sheath fluid; and

[0188] The frequency control unit acquires data about the state of the droplets at each frequency at each liquid temperature in the sheath fluid from images captured by the imaging unit, and controls the frequency of the drive voltage based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

[0189] [2] According to the particle separation device described in [1], the data is the data of the disconnection position of the droplet at each frequency.

[0190] [3] According to the particle separation device described in [2], the frequency control unit compares the break position of droplets at each liquid temperature of the sheath fluid.

[0191] [4] According to the particle separation device described in [3], the frequency control unit controls the frequency of the driving voltage based on the difference in the disconnection position of the droplets between the liquid temperatures of the sheath fluid.

[0192] [5] The particle separation device according to [4] further includes a determining unit, which determines the applicable frequency as an unstable frequency when the difference in the break position of the droplet is equal to or greater than a threshold.

[0193] [6] According to the particle separation device described in [5], the frequency control unit excludes unstable frequencies from the frequency options that can be used as the driving voltage.

[0194] [7] The particle separation apparatus according to any one of [4] to [6] further includes a determining unit, which determines the applicable frequency as a stable frequency when the difference in the break position of the droplets is less than a threshold.

[0195] [8] According to the particle separation device described in [7], the frequency control unit uses the frequency at which the difference in the disconnection position of the droplets is minimized as the frequency of the driving voltage.

[0196] [9] The particle separation device according to [6] further includes:

[0197] The charging unit charges a droplet containing a target particle.

[0198] In this process, droplets containing particles are formed by vibrations caused by the supply of a driving voltage based on a frequency other than an unstable frequency.

[0199]

[10] According to the particle separation device of [9], the liquid temperature control unit includes a temperature sensor arranged near the flow path, wherein the sample liquid containing particles and the sheath liquid are combined in the flow path.

[0200]

[11] The particle separation apparatus according to [9] or

[10] , wherein,

[0201] The imaging unit acquires images of the state of droplets containing particles, and

[0202] The particle separation device also includes a disconnection control unit, which controls the disconnection of droplets containing particles based on images of the state captured by the imaging unit.

[0203]

[12] The particle separation apparatus according to any one of [9] to

[11] , wherein the particles are biologically relevant particles.

[0204]

[13] A particle separation method, comprising:

[0205] The vibration step involves applying vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies;

[0206] The imaging step involves acquiring images of the fluid and droplets at the locations where the fluid is formed into droplets by vibration;

[0207] Liquid temperature control steps to control the liquid temperature of the sheath fluid; and

[0208] The frequency control step involves acquiring data on the state of the droplets at each frequency for each liquid temperature in the sheath fluid from images captured by the imaging unit, and controlling the frequency of the driving voltage based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

[0209]

[14] The particle separation method according to

[13] further includes a determination step, wherein when the difference in the break position of the droplet is equal to or greater than a threshold, the applicable frequency is determined as an unstable frequency.

[0210]

[15] The particle separation method according to

[14] further includes, after the determination step:

[0211] The charging step involves charging the droplet containing the target particle.

[0212] Droplets are formed by vibrations caused by the supply of a driving voltage based on a frequency other than the unstable frequency.

[0213]

[16] A program for causing a particle separation device to perform the following functions:

[0214] Vibrations are applied to a fluid containing sheath fluid by providing driving voltages based on multiple frequencies;

[0215] Images of the fluid and droplets are acquired at the locations where the fluid is formed into droplets through vibration;

[0216] Controlling the temperature of the sheath fluid; and

[0217] Data on the state of the droplets at each frequency for each liquid temperature in the sheath fluid is obtained from the captured images, and the frequency of the driving voltage is controlled based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

[0218] List of reference numerals

[0219] 1. Particle separation device

[0220] 11 Vibration Unit

[0221] 111 Vibration element

[0222] 12 imaging units

[0223] 121 Droplet Camera

[0224] 122 flashes

[0225] 13 Liquid temperature control unit

[0226] 14 Frequency Control Unit

[0227] 15. Determine the unit

[0228] 16A charging unit

[0229] 16b deflection plate

[0230] 16c Recycling Container

[0231] 17. Disconnect the control unit

[0232] 18 detection units

[0233] 181 Light Source

[0234] 19 Analysis Units

[0235] 20 storage units

[0236] 21 display units

[0237] 22 User Interface

[0238] P flow path

[0239] T chip

[0240] P11 Sample Liquid Flow Path

[0241] P12a, P12b Sheath fluid flow paths

[0242] P13 Mainstream Road

[0243] D droplets

[0244] BOP breakpoint

[0245] O-shaped opening

[0246] L-shaped liquid column.

Claims

1. A particle separation device, comprising: The vibration unit applies vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies; The imaging unit acquires images of the fluid and the droplets at the location where the fluid is formed into droplets by the vibration; A liquid temperature control unit controls the liquid temperature of the sheath fluid; as well as The frequency control unit acquires data about the state of the droplet at each frequency for each liquid temperature of the sheath fluid from the image captured by the imaging unit, and controls the frequency of the driving voltage based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

2. The particle separation device according to claim 1, wherein, The data refers to the location of the droplet's break at each frequency.

3. The particle separation device according to claim 2, wherein, The frequency control unit compares the disconnection position of droplets at each liquid temperature of the sheath fluid.

4. The particle separation device according to claim 3, wherein, The frequency control unit controls the frequency of the driving voltage based on the difference in the disconnection position of the droplets between the liquid temperatures of the sheath fluid.

5. The particle separation device according to claim 4 further includes a determining unit, wherein if the difference in the disconnection position of the droplet is equal to or greater than a threshold, the determining unit determines the applicable frequency as an unstable frequency.

6. The particle separation device according to claim 5, wherein, The frequency control unit excludes the unstable frequency from the frequency options that can be used as the driving voltage.

7. The particle separation device according to claim 4 further includes a determining unit, wherein if the difference in the disconnection positions of the droplets is less than a threshold, the determining unit determines the applicable frequency as a stable frequency.

8. The particle separation device according to claim 7, wherein, The frequency control unit uses the frequency at which the difference in the disconnection position of the droplet is minimized as the frequency of the driving voltage.

9. The particle separation device according to claim 6, further comprising: The charging unit charges a droplet containing a target particle. The droplets containing the particles are formed by vibrations caused by the supply of a driving voltage based on a frequency other than the unstable frequency.

10. The particle separation apparatus according to claim 9, wherein, The liquid temperature control unit includes a temperature sensor disposed near the flow path, in which the sample liquid containing the particles and the sheath liquid are combined.

11. The particle separation apparatus according to claim 9, wherein, The imaging unit acquires an image of the state of the droplet containing the particles, and The particle separation device further includes a disconnection control unit, which controls the disconnection of the droplet containing the particle based on an image of the state captured by the imaging unit.

12. The particle separation device according to claim 9, wherein, The particles in question are biologically relevant.

13. A particle separation method, comprising: The vibration step involves applying vibration to a fluid containing sheath fluid by providing a driving voltage based on multiple frequencies; The imaging step involves acquiring images of the fluid and the droplets at the locations where the fluid is formed into droplets by the vibration; A liquid temperature control step is used to control the liquid temperature of the sheath fluid; as well as The frequency control step involves acquiring data on the state of the droplet at each frequency for each liquid temperature in the sheath fluid from the image captured in the imaging step, and controlling the frequency of the driving voltage based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

14. The particle separation method according to claim 13 further includes a determining step, wherein if the difference in the break positions of the droplets is equal to or greater than a threshold, the applicable frequency is determined as an unstable frequency.

15. The particle separation method according to claim 14, further comprising, after the determining step, The charging step involves charging the droplet containing the target particle. in, The droplets are formed by vibrations caused by the supply of a driving voltage based on a frequency other than the unstable frequency.

16. A computer-readable storage medium storing a program that, when executed by a particle separation device, causes the particle separation device to perform the following functions: Vibrations are applied to a fluid containing sheath fluid by providing driving voltages based on multiple frequencies; Images of the fluid and the droplets are acquired at the locations where the fluid is formed into droplets by the vibration; Controlling the liquid temperature of the sheath fluid; as well as Data on the state of the droplets at each frequency for each liquid temperature of the sheath fluid is obtained from the captured images, and the frequency of the driving voltage is controlled based on the variation of the data accompanying the change in the liquid temperature of the sheath fluid.

Images