Particle sorting device, orifice unit for particle sorting device, and particle sorting method

Abstract

Provided is technology / technique that makes it possible to stabilize a droplet trajectory.　Provided are a particle sorting device and the like including: an irradiation unit that irradiates, with laser light, a portion of a flow path in which a fluid including particles is circulated; a detection unit that detects light generated by irradiation with the laser light; an orifice that is disposed on the flow path end and discharges the fluid; an electroconductive part disposed in the vicinity of the position where the fluid is made into droplets; and a charge part that applies a charge to the electroconductive part in accordance with optical data detected by the detection unit.

Description

[Technical Field]

[0001] The present technology relates to a particle sorting device, an orifice unit for a particle sorting device, and a particle sorting method, and more particularly to a particle sorting device, an orifice unit for a particle sorting device, and a particle sorting method that are capable of stabilizing droplet trajectories. [Background technology]

[0002] Currently, a technology called flow cytometry is used to analyze biological particles such as cells and microorganisms, as well as particles such as microbeads. Flow cytometry is an analytical technique in which particles are aligned and flowed into a fluid, and the light emitted from each particle is detected by irradiating the particles with light, thereby analyzing and separating the particles. The device used in flow cytometry is called a flow cytometer (also called a "cell sorter").

[0003] In a flow cytometer, a vibrating element is typically installed in a portion of the flow path through which particles encased in sheath fluid flow. This vibrating element vibrates a portion of the flow path, continuously breaking the fluid ejected from the flow path's orifice into droplets. Based on the detection signal obtained by irradiating light, the droplets containing the particles are given a positive (+) or negative (-) charge or are uncharged. They are then split by deflection plates according to their charge state, and the target particles are collected in their respective collection containers. The droplet groups deflected to the left or right by the positive or negative charge each follow a specific trajectory, appearing as linear, inclined streams. The uncharged droplet groups moving vertically downward are called the "center stream," while these linear, inclined streams are called "side streams."

[0004] It is important to efficiently and accurately charge the droplets using an appropriate method so that this side stream is properly guided to a collection container.In response to this, for example, Patent Document 1 discloses a technique for stabilizing droplets by controlling the drive voltage of a vibration element so that the distance between the tip of a droplet just before breakoff and the end of the satellite droplet just before it remains constant in a droplet observation image. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 115409 Brochure Summary of the Invention [Problem to be solved by the invention]

[0006] However, the technology for maintaining a constant sidestream trajectory is still insufficient, and further technological development is required.

[0007] Therefore, the main object of this technology is to provide a technology that can stabilize the droplet trajectory. [Means for solving the problem]

[0008] This technology first provides a particle sorting device having an irradiation unit that irradiates laser light onto a part of a flow path through which a fluid containing particles flows, a detection unit that detects light generated by the irradiation of the laser light, an orifice that is arranged at the end of the flow path and ejects the fluid, a conductive unit that is arranged near a position where the fluid is turned into droplets, and a charging unit that applies an electric charge to the conductive unit based on light data detected by the detection unit.

[0009] The present technology also provides an orifice unit for a particle sorting device, which has an orifice that is partially or entirely conductive, and a conductive portion that supports the orifice.

[0010] Furthermore, the present technology also provides a particle sorting method that includes an irradiation step of irradiating a part of a flow path through which a fluid containing particles flows with laser light, a detection step of detecting light generated by the irradiation with the laser light, and a charging step of applying an electric charge to a conductive part disposed near a position where the fluid is turned into droplets based on the light data detected by the detection part. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 10 illustrates the relationship between droplet period and correct timing of the charging signal. [Figure 2] FIG. 10 shows how droplets change near the break-off position and how side stream trajectories open and close when droplets with a droplet frequency of 100 kHz are left for 2000 seconds. [Figure 3] FIG. 1 is a diagram schematically illustrating an example of the configuration of a flow cell system. [Figure 4] FIG. 1 is a diagram schematically illustrating an example of a chip-type configuration. [Figure 5] FIG. 10 is a diagram schematically illustrating a configuration example of a charging method B. [Figure 6] 1A and 1B are diagrams schematically illustrating configuration examples of charging method A and charging method C. [Figure 7] FIG. 10 shows a comparison of the original signal waveform and the effective waveform at the orifice position when a pulse of ±175 V is applied to the metal sample liquid nozzle with the flow cell channel filled with sheath liquid. [Figure 8] 1 is a diagram schematically illustrating a configuration example of a first embodiment of a particle sorting device 1 according to the present technology. [Figure 9] FIG. 2 is a diagram schematically showing another configuration example of the first embodiment of the particle sorting device 1 according to the present technology. [Figure 10] FIG. 2 is a diagram schematically illustrating an example of the configuration of a ground electrode. [Figure 11] FIG. 10 is a diagram showing an example of the configuration of an optical system around a droplet formation unit in the case of a flow cell system. [Figure 12] 1A to 1C are diagrams schematically showing examples of the orifice O and the conductive portion R. [Figure 13]1D to 1F are diagrams schematically showing examples of the orifice O and the conductive portion R. [Figure 14] GI are diagrams showing examples of the orifice O and the conductive portion R. [Figure 15] 2 is a diagram schematically illustrating an example of the configuration of an orifice O according to a first embodiment of the orifice unit U. FIG. [Figure 16] 1 is a diagram schematically illustrating a configuration example of a first embodiment of an orifice unit U. FIG. [Figure 17] FIG. 10 is a diagram schematically illustrating a configuration example of a second embodiment of the orifice unit U. [Figure 18] FIG. 10 is a diagram schematically illustrating a configuration example of a third embodiment of the orifice unit U. [Figure 19] FIG. 10 is a diagram schematically illustrating a configuration example of a fourth embodiment of the orifice unit U. [Figure 20] FIG. 10 is a diagram schematically illustrating a configuration example of an embodiment in the case of a chip system. [Figure 21] FIG. 10 is a diagram showing the results of comparing the waveforms of charging signals in an example and a comparative example. [Figure 22] FIG. 10 is a diagram showing the results of a comparison of the relationship between the side stream deflection distance and the charge signal phase in an example and a comparative example. [Figure 23] FIG. 10 is a diagram showing a comparison result of charge waveforms regarding correction of charge signals. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings. The embodiment described below is an example of a typical embodiment of the present technology, and is not to be construed as narrowing the scope of the present technology. The description will be made in the following order. 1. Overview of this technology 2. First Embodiment (Particle Sorting Apparatus 1) (1) Flow path P (2) Irradiation unit 11 (3) Detection unit 12 (4) Orifice O (5) Conductive part R (6) Charged part 13a (7) Deflection plate 13b, collection container 13c (8) Vibration unit 14 (9) Imaging unit 15 (10) Break-off control unit 16 (11) Analysis section 17 (12) Storage section 18 (13) Display section 19 (14) User Interface 20 (15) Other 3. Examples of orifice O and conductive part R (1) Example of flow cell system (2) Example of chip type (3) Orifice unit U for particle sorting device (3-1) First embodiment of orifice unit U (3-2) Second embodiment of orifice unit U (3-3) Third embodiment of orifice unit U (3-4) Fourth embodiment of orifice unit U (4) Chip-type embodiment 4. Second embodiment (particle sorting method)

[0013] 1. Overview of this technology

[0014] This technology is used in a device that irradiates light onto particles aligned in a flow path, detects the light emitted from each particle, and based on the detection signal, gives a positive (+) or negative (-) charge to droplets containing the particles using a counter electrode, or leaves them uncharged, and splits them into individual droplet trajectories using a deflection plate to recover the target particles.The technology uses an appropriate method to efficiently and accurately charge droplets in order to maintain a constant side stream trajectory that carries the particles to a recovery container.

[0015] Droplets containing the target particles are charged by contacting an electrode with the conductive sheath liquid in the droplet formation unit and applying a pulse signal of positive or negative polarity to the electrode depending on the deflection direction. The charging signal is transmitted to the tip of the liquid column through the sheath liquid, and an amount of charge proportional to the voltage immediately before the droplet breaks off is applied. In this case, the width of the charging pulse is generally the same as one droplet cycle T (for example, T = approximately 10 μsec for droplets with a droplet frequency of 100 kHz), and the voltage is approximately ±100 to 200 V.

[0016] Here, to stabilize the trajectory of the side stream consisting of droplets containing the target particles, accurate charging is required so that each droplet is given a uniform amount of charge. As mentioned above, the droplets are charged at the moment they break off from the liquid column, so it is essential to adjust the timing of the droplet breakoff (hereafter referred to as "breakoff") and the charging pulse and apply the maximum voltage. If this timing is not adjusted properly, the droplets will not be charged sufficiently, the deflection angle will narrow in proportion to the amount of charge, and the side streams will close inward.

[0017] Typically, the charging pulse has a time duration (T) equal to one droplet cycle. Therefore, the timing must be adjusted so that the breakoff time of the droplet containing the target particle falls within the charging pulse duration (T). However, the actual charging pulse has a signal rise time (Tr) and fall time (Tf). Therefore, the effective pulse duration (Te) at which the maximum voltage (Vtop) is obtained is calculated by subtracting these times from T, i.e., Te = T - (Tf + Tr). For example, if the droplet frequency is 100 kHz, the period (T) is 10 μsec. If both Tr and Tf are 3 μsec, Te is halved to 4 μsec. Therefore, simply, this Te value can be considered the allowable margin for time variation in breakoff. Figure 1 shows the relationship between the droplet period and the correct timing of the charging signal.

[0018] The variation in the break-off timing of the droplets can be observed in detail, for example, by illuminating the droplets with a light source that flashes in synchronization with the piezo drive signal and obtaining a strobe image from a droplet observation camera. Figure 2 shows how the droplet changes near the break-off position and the side stream trajectory opens and closes when a droplet with a droplet frequency of 100 kHz is left standing for 2000 seconds.

[0019] In the example shown in Figure 2, the phase of the charging pulse is synchronized with the droplet so that the side stream opens to its maximum angle at the start of observation. As a result, the timing of breakoff advances over time, and changes can be seen, particularly in the length and position of the satellite droplets located between the main droplets. After 2000 seconds, the breakoff timing has advanced by approximately one droplet cycle (i.e., T), so the side stream returns to its maximum angle again, but the upper droplet, one droplet away, is deflected, rather than the lower droplet that should be charged.

[0020] From the above, fluctuations in the droplet breakoff timing are a direct cause of disturbances in the sidestream trajectory, and therefore require strict control. In response to this, for example, in Patent Document 1, droplets are photographed using a strobe light synchronized with the frequency, and feedback control of the piezo drive voltage is performed based on the strobe image information to prevent changes near the BOP. However, even with feedback control, fluctuations in the droplet break-off timing cannot be maintained at zero at all times, and fluctuations of approximately ±0.1 to 0.2 T may remain. Therefore, it is considered important to maximize the effective pulse width (Te) at which the maximum voltage (Vtop) of the charging pulse is obtained in order to ensure the stability of the sidestream trajectory.

[0021] Here, one method for reducing the rise time Tr and fall time Tf of a charging pulse with a time width T and ensuring a wide effective pulse width Te is to optimize the position of the electrode that supplies the charging signal.

[0022] The electrode that applies the charging signal to the sheath liquid should be placed as close to the BOP as possible. This is because it takes a certain amount of time for electrons and ions to move from the electrode to the breakoff position at the tip of the liquid column after voltage application. The voltage V applied to the droplet after the voltage V0 is applied as a function of the elapsed time t is expressed as V = V0 × (1 − exp(−t / τ)). Here, the time constant τ is proportional to r × C, which is the product of the resistance r between the electrode and the BOP and the capacitance C between the sheath liquid column and the ground electrode. A smaller time constant τ reduces the rise / fall time and increases the effective pulse width Te. Therefore, it is desirable to reduce the resistance r between the electrode and the BOP. This resistance r is determined by the sheath liquid (electrical resistivity: approximately 0.2 Ω·m) present between the electrode and the BOP. Therefore, shortening the distance between the electrode and the BOP is the solution.

[0023] Here, the location of the charge signal electrodes in a conventional cell sorter will be described. The droplet formation unit, including the electrode, typically consists of a flow path where the sheath liquid and sample flow merge to form a laminar flow, a piezoelectric excitation unit that vibrates the liquid at a desired frequency, a detection unit where laser light is irradiated onto particles in a linear flow path, and an orifice that ejects light from the particles and the liquid column. There is also a type known as the "Jet in Air" method, in which laser light is irradiated onto particles in the liquid column after the sheath liquid containing the particles is ejected from the orifice. Commercially available products can be broadly classified into the following two types, but the basic configuration described above is the same. - Flow cell system in which the fluidic system is fixed and only the nozzle at the tip is replaceable (see Figure 3) The entire flow path system, including the orifice, is integrated and replaceable using a chip (see Figure 4).

[0024] The charging signal is applied to the sheath liquid via an electrode within the droplet formation unit. A ground electrode, connected to ground (GND), is also required within 1 mm of the BOP. Although the ground electrode and the sheath liquid are not in contact, they are grounded at the end of the liquid column, and charging occurs in proportion to the potential difference between the signal and the ground electrode. Because electrical insulation between the two electrodes is important, the main components of the droplet formation unit, whether it is the flow cell type shown in Figure 3 or the chip type shown in Figure 4, must be made of insulating materials. Therefore, there are essentially no locations within the droplet formation unit where the sheath liquid comes into contact with conductive materials.

[0025] Therefore, conventionally, the sheath liquid has been charged by wiring a charge signal to a metal joint at the sheath liquid tube attachment (charging method A; see Figure 6) or by inserting a metal wire into the flow path (charging method B; see Figure 5). Alternatively, Japanese Patent Application Laid-Open No. 2010-54492 proposes a technique in which a sample liquid nozzle for merging particle-containing sample liquid with the sheath liquid is formed from a metal microtube, and a charge signal is applied to the metal microtube (charging method C; see Figure 6).

[0026] However, with these methods, the sheath liquid is charged just before it meets the sample liquid and forms a laminar flow, making it difficult to move the charging position closer to the BOP. For example, extending the metal wire to a point where laminar flow is already formed could disrupt the laminar flow due to vibrations or other factors. Furthermore, the cross-section of the flow channel narrows and becomes smaller as it moves from the inlet to the orifice, which has an opening diameter of approximately 0.1 mm. After the linear flow channel inside the cuvette, the diameter narrows to 0.3 mm or less, making it physically more difficult to place the metal wire closer to the orifice.

[0027] Thus, in an actual cell sorter, the position of the electrode that charges the sheath liquid is limited to the first half of the droplet formation unit, i.e., just before the sheath liquid and sample liquid merge to form a laminar flow. However, because the distance from the charging position to the BOP is approximately 40–50 mm, it takes a certain amount of time for the charge to move to the BOP. As a result, the effective charging waveform becomes dull relative to the amplifier output signal. As the effective pulse width Te at which the maximum voltage Vtop is obtained decreases, the margin for charging timing decreases, resulting in a destabilized sidestream trajectory. This tendency becomes more pronounced as the droplet frequency increases, i.e., the charging pulse width becomes shorter. This tendency is explained in detail below.

[0028] In the configuration shown in Figure 6, a sample liquid nozzle was fabricated from metal and a charging signal cable was wired according to charging method C described above. The distance from the bottom of the metal sample liquid nozzle to the orifice was 28 mm in total, including the 0.2 mm square x 15 mm long linear flow path in the cuvette directly above the orifice. Next, a metal plate was attached to the orifice position, and the oscilloscope probe was brought into contact with it to measure the effective charging pulse waveform. Figure 7 shows a comparison of the original signal waveform (AMP output waveform) and the effective waveform at the orifice when a ±175V pulse is applied to the metal sample liquid nozzle with the flow cell channel filled with sheath liquid. Figure 7A shows the results when the pulse width T1 is set to 50 μsec (equivalent to a 20 kHz droplet), and Figure 7B shows the results when the pulse width T2 is set to 10 μsec (equivalent to a 100 kHz droplet).

[0029] When T1 = 50 μsec, the rise time of the AMP output waveform increased slightly, but the waveform was maintained with almost no degradation, posing no problem. On the other hand, when T2 = 10 μsec, a pulse width shorter than T1, the rise time became similar to T2. As a result, the maximum voltage (Vtop) of the charging pulse became so sluggish that it was almost zero, and the voltage amplitude also decreased by 6%. If a sidestream were formed under these conditions, the timing at which the deflection angle reached its maximum became pinpointed, and the deflection angle decreased with a timing fluctuation of ±0.1 to 0.2 T. This made it difficult to maintain a constant sidestream trajectory, and the maximum deflection angle also became insufficient compared to its intended value.

[0030] In view of the above, there is a need to provide a technology that can suppress deterioration of the charging pulse waveform, particularly in high-frequency droplets, and charge droplets as faithfully as possible to the charging pulse output waveform, thereby ensuring as much margin as possible for the charging timing and stabilizing the side stream trajectory for a long period of time.

[0031] 2. First Embodiment (Particle Sorting Apparatus 1)

[0032] Fig. 8 shows a configuration example of a first embodiment of the particle sorting device 1 according to the present technology, and Fig. 9 shows another configuration example of the first embodiment of the particle sorting device 1 according to the present technology. 8 and 9 includes at least an irradiation unit 11, a detection unit 12, an orifice O, a conductive unit R, and a charging unit 13a. The particle sorting device 1 may also include a flow path P, a deflection plate 13b, a collection container 13c, a vibration unit 14, an imaging unit 15, a break-off control unit 16, an analysis unit 17, a memory unit 18, a display unit 19, a user interface 20, and the like, as needed.

[0033] (1) Flow path P

[0034] A fluid containing particles flows through the flow path P. If necessary, a sample liquid containing particles and a sheath liquid that flows so as to enclose the sample liquid may also flow through the flow path P. In this case, the flow path P may be configured to form a flow in which the particles are aligned in a substantially straight line. The flow path P may be provided in advance in the particle sorting device 1, but it is also possible to install a commercially available flow path or a disposable microchip provided with a flow path.

[0035] The shape of the flow channel P is not particularly limited and can be freely designed as appropriate. For example, the flow channel is not limited to the flow channel formed in a two-dimensional or three-dimensional substrate made of plastic, glass, or the like shown in Fig. 4, but a flow channel such as that used in a conventional flow cytometer shown in Fig. 3 can also be used.

[0036] The width, depth, and cross-sectional shape of the flow channel P are not particularly limited and can be freely designed as appropriate. For example, a micro flow channel with a width of 1 mm or less can also be used in the particle sorting device 1.

[0037] In the present technology, the term "particles" may broadly include biological particles such as cells, microorganisms, and ribosomes, as well as synthetic particles such as latex particles, gel particles, and industrial particles. In addition, in the present technology, the particles may be contained in a fluid such as a liquid sample.

[0038] Bio-related particles may include chromosomes, ribosomes, mitochondria, organelles (cell organelles), and the like that make up various cells. Cells may include animal cells (e.g., blood cells) and plant cells. Microorganisms may include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Bio-related particles may also include bio-related polymers such as nucleic acids, proteins, and complexes thereof. Industrial particles may be, for example, organic or inorganic polymer materials, metals, etc. Organic polymer materials may include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials may include glass, silica, magnetic materials, etc. Metals may include gold colloid, aluminum, etc. Although the shape of these particles is generally spherical, in the present technology, they may be non-spherical, and there are no particular limitations on their size, mass, etc. In the present technology, the particles are preferably biologically relevant particles, and cells are particularly preferred.

[0039] The particles may be labeled with one or more dyes such as fluorescent dyes, etc. In this case, examples of usable fluorescent dyes include Cascade Blue, Pacific Blue, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Propidium iodide (PI), Texas Red (TR), Peridinin chlorophyll protein (PerCP), Allophycocyanin (APC), 4',6-Diamidino-2-phenylindole (DAPI), Cy3, Cy5, Cy7, and Brilliant Violet (BV421).

[0040] (2) Irradiation unit 11

[0041] The irradiation unit 11 irradiates a part of the flow path P through which the fluid containing particles flows with laser light. Specifically, the irradiation unit 11 irradiates the particles flowing in the main flow path P13 while being aligned substantially in a line at the center of the three-dimensional laminar flow with laser light.

[0042] The irradiation unit 11 includes one or more light sources. When the irradiation unit 11 includes multiple light sources, the laser beams emitted from the multiple light sources may be combined, and the combined laser beam may be irradiated onto the particles. Furthermore, the irradiation unit 11 may be configured to irradiate the laser beams from the multiple light sources at different positions in the flow direction of the fluid. In the present technology, the multiple light sources may emit laser beams having the same wavelength or different wavelengths.

[0043] The type of laser light irradiated from the irradiation unit 11 is not particularly limited, but examples include a semiconductor laser, an argon ion (Ar) laser, a helium-neon (He-Ne) laser, a dye laser, a krypton (Cr) laser, and a solid-state laser that combines a semiconductor laser with a wavelength conversion optical element, and two or more of these can also be used in combination.

[0044] The irradiation unit 11 may also include a light-guiding optical system for guiding the laser light to a predetermined position. The light-guiding optical system may include optical components such as a beam splitter group, a mirror group, and an optical fiber. The light-guiding optical system may also include a lens group for focusing the combined excitation light, and may include, for example, an objective lens.

[0045] (3) Detection unit 12

[0046] The detection unit 12 detects light generated by the irradiation of laser light by the above-mentioned irradiation unit 11. Specifically, the detection unit 12 detects fluorescence and scattered light (for example, forward scattered light, back scattered light, side scattered light, Rayleigh scattering, Mie scattering, etc.), which are light to be measured, generated from the particles by irradiating the particles with laser light.

[0047] The detection unit 12 includes at least one photodetector that detects the light to be measured. The photodetector includes one or more light-receiving elements and may have, for example, a light-receiving element array. The photodetector may have, as the light-receiving element, one or more photodiodes such as a PMT (photomultiplier tube), an APD (avalanche photodiode), or an MPPC (multi-pixel photon counter). In this case, the photodetector may be, for example, a PMT array in which a plurality of PMTs are arranged in a one-dimensional direction. The detection unit 12 may also include an imaging element such as a CCD (charge coupled device) or a CMOS (complementary metal-oxide-semiconductor).

[0048] The detection unit 12 includes a signal processing unit such as an A / D converter that converts the electrical signal obtained by the photodetector into optical data (digital signal). The optical data obtained by the conversion by the signal processing unit is transmitted to the analysis unit 17 described below. Examples of the optical data include optical data including fluorescence data, and more specifically, light intensity data of light including fluorescence (for example, feature quantities such as area, height, and width).

[0049] The detector 12 may also include a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system may include, for example, a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter.

[0050] (4) Orifice O

[0051] The orifice O is disposed at the end of the flow path P and discharges a fluid containing particles. The specific configuration of the orifice O will be described later in "3. Examples of configurations of the orifice O and the conductive portion R."

[0052] (5) Conductive part R The conductive portion R is disposed at a position where the fluid containing particles turns into droplets, i.e., near the BOP. The specific shape of the conductive portion R will be described later in "3. Examples of shapes of the orifice O and conductive portion R."

[0053] In the present technology, the conductive portion R is preferably formed from a conductive material, and examples of the conductive material include metals such as stainless steel and titanium; conductive resins filled with conductive fillers such as carbon or metal powder or fibers; and non-conductors (e.g., resins, ceramics, etc.) whose surfaces are made conductive by vapor deposition or sputtering of metals such as gold, platinum, nickel, and chromium.

[0054] The conductive portion R is preferably arranged downstream of the optical detection region P14, which is the region irradiated with the laser light, in the flow direction of the fluid containing particles, which allows it to be easily arranged near the BOP.

[0055] This technology applies a charging signal to the orifice O closest to the BOP (droplet breakup point) when particles are ejected as a liquid column L from the orifice O in a cell sorter and eventually break up into droplets. Conventionally, charging was performed by installing a charging electrode near the entrance to the droplet formation unit. However, with this technology, the BOP is much closer than the installation position of the charging electrode, reducing the travel time of the charge or ions. This reduces the rise / fall time of the effective charging wave form, and the charging AMP output waveform is applied to the droplets with almost no degradation. As a result, the following effects are obtained regarding the sidestream trajectory.

[0056] First, the effective charging signal maintains the maximum voltage (Vtop) for a maximum period (Te), which maximizes the margin for the charging timing to obtain the maximum deflection angle. As a result, the stability of the sidestream trajectory is ensured even when, for example, a slight fluctuation occurs in the droplet breakup timing due to a change in the sheath liquid temperature.

[0057] Furthermore, with conventional charging methods, the pulse rise / fall time exceeds the charging signal pulse width, preventing the charging pulse from reaching the maximum voltage Vtop and resulting in an inability to obtain the intended deflection angle.In contrast, with the orifice charging method of this technology, such degradation does not occur, and the intended deflection angle can be obtained for the charging signal output voltage.

[0058] Furthermore, because the specified charging signal is applied to the droplets without degradation, it becomes possible to make fine corrections to the charging voltage for each droplet in accordance with various sorting patterns, making it easier to focus the side stream trajectory within a certain range regardless of the pattern.

[0059] These effects become more pronounced as the droplet frequency increases, and are therefore particularly useful when deflecting droplets with a frequency of preferably 50 kHz or higher, more preferably around 100 kHz.

[0060] (6) Charged part 13a

[0061] The charging unit 13a applies an electric charge to the conductive unit R based on the optical data detected by the detection unit 12. Specifically, the charging unit 13a applies a charging signal to the conductive unit R as needed, thereby applying a positive or negative electric charge to the desired droplet D.

[0062] The charging unit 13a preferably includes a charging electrode for applying a charging signal and a ground electrode disposed near the BOP. The ground electrode can be, for example, a U-shaped metal member surrounding the liquid column L, as shown in Fig. 10, and positioned by adjusting a movable stage or the like so that the metal member approaches the BOP by approximately 0.5 mm. The ground side of the charging signal line is then connected to the electrode made of the metal member.

[0063] The charging unit 13a may also correct the charge amount of the droplets. Specifically, the charging unit 13a applies a corrective voltage to the charge signal, a process known as defanning. This prevents the zero-charge droplet group, i.e., the center stream, from spreading, allowing the waste container to be narrower, thereby narrowing the deflection angle of the side streams. Furthermore, when sorting at a high frequency (e.g., when sorting with almost no intervening zero-charge droplets) or when charging is performed continuously in the same direction, the side streams are also affected by the charge of droplets adjacent to the front, and defanning can be used to prevent the side streams from splitting.

[0064] (7) Deflection plate 13b, collection container 13c

[0065] The deflection plate 13b controls the direction of travel of the desired droplets D based on the presence or absence of an electrical force and the magnitude of the force, and guides the droplets D to a predetermined collection container 13c.

[0066] Specifically, the deflection plates 13b deflect the direction of travel of each droplet D in the fluid stream by an electrical force acting between the positive or negative charge applied to the droplet D and the deflection plates 13b, guiding the droplets D to a predetermined collection container 13c. The deflection plates 13b are arranged opposite each other across the fluid stream. There are no particular limitations on the deflection plates 13b, and conventionally known electrodes or the like can be used. Different positive or negative voltages are applied to the deflection plates 13b, and when the charged droplets D pass through the electric field formed thereby, an electrical force (Coulomb force) is generated, and each droplet D is attracted toward one of the deflection plates 13b.

[0067] A plurality of collection containers 13c may be arranged in a line in the direction in which the deflection plates 13b face each other. The type of collection container 13c is not particularly limited, and examples thereof include plastic tubes and glass tubes. The number of collection containers 13c is also not particularly limited, but FIGS. 8 and 9 show an example in which three collection containers 13c are installed. The collection containers 13c may be replaceably installed in a collection container container (not shown). Specifically, for example, the collection containers 13c may be arranged on a Z-axis stage (not shown) configured to be movable in a direction perpendicular to the discharge direction of the droplets D from the orifice O and the facing direction of the deflection plates 13b.

[0068] (8) Vibration unit 14

[0069] The vibration unit 14 applies vibration to the fluid by supplying a driving voltage based on one or more frequencies. This allows the fluid to be continuously broken into droplets and generate a fluid stream. The frequency may be within a frequency range specified by a user.

[0070] The vibration is applied, for example, by a vibration element. The vibration element is not particularly limited, and a conventionally known one can be used, for example, a piezoelectric element. When a chip is used as the flow path P, the vibration element is preferably provided near the orifice O of the chip.

[0071] In the flow cell system shown in Figure 3, sheath and sample liquids are first injected into a conical vessel. The conical vessel is placed with its apex pointing vertically downward, and a tube for introducing sheath liquid is connected to the upper side. The top of the conical vessel is open, and a vibrating element is attached, sealed with an O-ring. The sample liquid is injected vertically from above the vessel. The vibrating element and piston are annular, and piping passes through their central hole. The conical vessel narrows at the bottom and connects to a cuvette section, which has a main channel (linear channel) P13 formed inside. A laminar flow is formed within the conical vessel, with the sheath liquid surrounding the sample liquid. The laminar flow continues into the cuvette section, where detection is performed by irradiating the main channel P13 with laser light. A removable outlet nozzle is installed at the end of the main channel P13, and the nozzle slopes downwards, narrowing continuously from the cuvette outlet to the orifice O. The sheath and sample liquids are subjected to minute vibrations in the forward and backward directions relative to the flow by a vibration element attached directly above the conical vessel. The liquid column L ejected from the orifice O advances vertically downward, expanding the formed constriction at the same frequency as the vibrations caused by the vibration element, and breaks down into droplets at the BOP, located 10-20 mm from the orifice O.

[0072] An example of the configuration of the optical system around the droplet formation unit in the case of the flow cell system is shown in Figure 11. Around the droplet formation unit, there are provided a droplet camera 151 and a strobe 152 that constitute the imaging unit 15, an irradiation unit 11, a forward scattered light detector 121 and a side fluorescent light detector 122 that constitute the detection unit 12, and the like.

[0073] In the chip system shown in Figure 4, the sheath liquid inlet and sheath liquid flow path P12, the sample liquid inlet and sample liquid flow path P11, the main flow path (linear flow path) P13 where these flow paths converge and through which light is irradiated, and the orifice O are all integrated and replaceable. The sample liquid flow path P11 is linearly arranged in the center, and the sheath liquid flow path P12 branches off to the left and right from the inlet, surrounding the sample liquid flow path P11. Eventually, the three flow paths converge at one point to form the main flow path P13. This creates a laminar flow between the sheath liquids, and the sample liquid proceeds to the optical detection region P14, where it is detected by laser light irradiation. Furthermore, an annular flow path P15 is arranged at the outermost periphery and connected to the main flow path P13 on both sides. This flow path is connected to an external pump and used to remove air bubbles generated within the flow path. In this case, when a portion of the substrate surface forming the chip is vibrated by a vibration element, droplets D are formed from the liquid column L ejected from the orifice O. Alternatively, the sheath liquid may be vibrated directly before the entrance of the tip.

[0074] (9) Imaging unit 15

[0075] The imaging unit 15 captures images of the fluid and droplets D before they are broken down into droplets in the BOP.

[0076] The imaging unit 15 may be, for example, a droplet camera 151 such as a CCD camera or a CMOS sensor. The droplet camera 151 may be disposed between the orifice O and the deflection plate 13b at a position where it can capture an image of the droplet D. The droplet camera 151 may also adjust the focus of the captured image of the droplet D. A light source that illuminates the imaging area in the droplet camera 151 may be, for example, a strobe 152. The imaging unit 15 may also capture a phase image at a certain time, or may continuously capture the images within a certain period. The "certain period" referred to here is not particularly limited and may be one period or multiple periods. In the case of multiple periods, the periods may be continuous or discontinuous in time.

[0077] The image captured by the imaging unit 15 is displayed on the display unit 19, which will be described later, and can be used by the user to check the formation status of the droplets D (for example, the size, shape, spacing, etc. of the droplets D). The strobe 152 may be controlled by the break-off control unit 16, which will be described later. The strobe 152 is composed of, for example, an LED for capturing images of the droplets D and a laser (for example, a red laser light source) for capturing images of the particles, and can be switched by the break-off control unit 16, which will be described later, depending on the purpose of capturing the images, etc. The specific structure of the strobe 152 is not particularly limited, and conventionally known circuits and / or elements can be used.

[0078] (10) Break-off control unit 16

[0079] The breakoff control unit 16 controls the breakoff of the droplet D containing the target particle based on an image of the state of the droplet D containing the particle acquired by the above-described imaging unit 15. Specifically, based on the timing at which the droplet D containing the particle breaks off, which is specified from multiple droplet observation images acquired by the imaging unit 15, the breakoff control unit 16 adjusts the drive voltage of the vibration element to control the bonding state between the droplet D and the liquid column L and / or the distance between the droplet D and the liquid column L, and the breakoff position of the droplet D to be maintained constant. In this way, by constantly applying feedback to the drive voltage to adjust the droplet, it is possible to prevent the droplet D from becoming unstable after sorting begins.

[0080] (11) Analysis section 17

[0081] The analysis unit 17 is connected to the detection unit 12, the imaging unit 15, etc., and performs analysis based on the optical data acquired by the detection unit 12, the image acquired by the imaging unit 15, etc.

[0082] Specifically, the analysis unit 17 calculates the characteristic quantities of each particle based on the optical data acquired by the detection unit 12. For example, the analysis unit 17 calculates the characteristic quantities such as particle size, shape, and internal structure from the detection values ​​of the received fluorescence and scattered light. Furthermore, the analysis unit 17 makes a sorting decision based on the calculated characteristic quantities and the sorting conditions received from the user interface 20 (described later), and generates a sorting control signal. By applying a charging signal to the charging unit 13a described above based on the sorting control signal, specific types of particles can be sorted and collected. Furthermore, the analysis unit 17 analyzes or calculates data regarding the state of the droplet D based on the image acquired by the imaging unit 15.

[0083] In the present technology, the analysis unit 17 may be included in a housing in which the detection unit 12 and the like are provided, or may be located outside the housing. Furthermore, the analysis unit 17 is not essential to the particle sorting device 1 according to the present embodiment, and an external analysis device or the like may also be used. Furthermore, the analysis unit 17 may be connected to each part of the particle sorting device 1 via a network.

[0084] (12) Storage section 18

[0085] The memory unit 18 stores all kinds of information, such as the optical data detected by the detection unit 12, the feature values ​​of each particle calculated by the analysis unit 17, the generated sorting control signal, and the sorting conditions input via the user interface 20.

[0086] In the present technology, the storage unit 18 may be included in a housing in which the detection unit 12 and the like are provided, or may be located outside the housing. Furthermore, the storage unit 18 is not essential to the particle sorting device 1 according to the present embodiment, and an external storage device (e.g., a hard disk) or the like may also be used. Furthermore, the storage unit 18 may be connected to each part of the particle sorting device 1 via a network.

[0087] (13) Display section 19

[0088] The display unit 19 can display all sorts of information, for example, the feature amount of each particle calculated by the analysis unit 17 can be displayed as a histogram or the like. It may also display images captured by the imaging unit 15.

[0089] The display unit 19 is not essential to the particle sorting device 1 according to this embodiment, and an external display device (for example, a display, a printer, a personal digital assistant, etc.) can also be used. The display unit 19 may be connected to each part of the particle sorting device 1 via a network.

[0090] (14) User Interface 20

[0091] The user interface 20 is a component for user operation. The user can input various data via the user interface 20 and access and control each part of the particle sorting device 1. Specifically, for example, via the user interface 20, the user can set an area of ​​interest for a histogram or the like displayed on the display unit 19 and determine sorting conditions and the like.

[0092] The user interface 20 is not essential to the particle sorting device 1 according to this embodiment, and an external operating device (e.g., a mouse, a keyboard, a personal digital assistant, etc.) can also be used. The user interface 20 may be connected to each part of the particle sorting device 1 via a network.

[0093] (15) Other

[0094] The functions performed by each part of the particle sorting device 1 according to the present technology can be stored as a program in a general-purpose computer, a control unit including a CPU, etc., and hardware resources including a recording medium (e.g., non-volatile memory (e.g., USB memory, etc.), HDD, CD, etc.), etc. The functions can also be realized by a server computer or cloud connected via a network.

[0095] 3. Examples of orifice O and conductive part R

[0096] Examples of the orifice O and the conductive portion R will be described below with reference to the drawings. 12 to 14 schematically show various examples of the orifice O and the conductive portion R. Fig. 12 and Fig. 13 show examples of the flow cell type, and Fig. 14 shows an example of the chip type.

[0097] (1) Example of the flow cell system

[0098] 12A shows an example in which a metal orifice O and a metal conductive part R supporting the orifice O are abutted against the end of a cuvette flow channel through which a particle-containing fluid flows, via a sealing member such as an O-ring. Also, FIG. 12B differs from the example shown in FIG. 12A in that the orifice O and the conductive part R are made of resin that has been made conductive with a conductive filler. Furthermore, FIG. 12C differs from the example shown in FIG. 12A in that the orifice O and the conductive part R are made of non-conductors such as resin or ceramic, and that portions of the conductive part R are made conductive by evaporating or sputtering metal onto them. The embodiments shown in Figures 12B and 12C can be manufactured more cheaply than the embodiment shown in Figure 12A, and therefore the orifice O or the conductive part R supporting the orifice O can be replaced.

[0099] Figure 13D shows an example in which the orifice O is made of a non-conductor such as resin or ceramic, and a conductive part R made of metal is provided between the end of the cuvette flow path and the orifice O, and the conductive part R is adhered so as to abut against the orifice O. In the example shown in Figure 13D, the orifice O itself can be made of a non-conductor, which broadens the options for materials and manufacturing methods. In addition, in this case, the orifice O can be manufactured inexpensively, and therefore can be made disposable.

[0100] 13E differs from the embodiment shown in FIG. 12A in that the orifice O and the conductive part R that supports the orifice are held down by a metal cover. The cover can fix the orifice O and the conductive part R, and it is also possible to supply power to the orifice O and the conductive part R through the cover. In this embodiment, the cover is not limited to metal, and may be made of other conductive materials.

[0101] 13F differs from the embodiment shown in FIG. 12A in that it includes a cover, a positioning mechanism for attachment to the end of the flow path, and a metal contact probe. Note that in this embodiment, the cover and the positioning mechanism do not necessarily have to be conductive. The contact probe may function as an elastic contact point using a spring or the like, which allows for easy installation of the orifice O at the end of the flow path in conjunction with the positioning mechanism. Note that in this embodiment, the contact probe may be made of not only metal but also other conductive materials.

[0102] 12 and 13, the conductive part R may have a connection part R1 that connects to the charging part 13a, or the charging part 13a may be directly connected to the conductive part R. Furthermore, if the conductive part R is replaceable, the conductive part R may have a holding part R2 that the user holds when replacing it.

[0103] (2) Example of chip type

[0104] 14G shows an example in which the entire chip is made of a conductive material. In this example, by making part or all of the orifice O conductive, the orifice O itself functions as a conductive part R. This example is also effective when employing the "Jet-in-Air method," in which the particles are irradiated with laser light in the liquid column after the sheath liquid containing the particles is ejected from the orifice O.

[0105] 14H shows an example in which the optical detection region P14 of the chip is made of an optically detectable material such as quartz or transparent resin, and the other parts are made of a conductive material. In this example, the orifice O itself also functions as the conductive part R. This allows optical detection to be performed within the chip.

[0106] Figure 14I shows an example in which the entire chip is made of a material that can be optically detected, and a thin metal film is formed near the orifice O by vapor deposition, sputtering, or the like. In this example, the orifice O itself functions as the conductive part R. This allows chips including the orifice O to be manufactured inexpensively, and chips including an orifice O with a portion that is conductive can also be disposable.

[0107] (3) Orifice unit U for particle sorting device

[0108] The present technology also provides an orifice unit U for a particle sorting apparatus, which has an orifice O that is partially or entirely conductive, and a conductive portion R that supports the orifice O. Based on the embodiment examples shown in Figs. 12 and 13 described above, an embodiment of the orifice unit U according to the present technology will be described in detail below with reference to the drawings.

[0109] (3-1) First embodiment of orifice unit U

[0110] FIG. 15 shows an orifice O according to a first embodiment of an orifice unit U for a particle sorting apparatus. The orifice O shown in FIG. 15 is a chip type orifice, entirely made of a conductive material. Specifically, for example, as shown in FIG. 15A, an opening is machined in the center of a chip with an outer diameter of 5 mm and a thickness of 1.5 mm. As shown in FIG. 15B, the orifice O has a 1.2 mm long circular channel with a diameter of 0.3 mm, which is continuous with the main channel (straight channel) P13 in front of it. A 0.2 mm long sloped section narrowing from 0.3 mm to 0.07 mm is inserted beyond the channel, and a 0.1 mm long nozzle section with a diameter of 0.07 mm is formed at the end.

[0111] FIG. 16 also shows the orifice O and conductive portion R according to the first embodiment. FIG. 16A shows the orifice O attached to the conductive portion R, FIG. 16B shows the state before the orifice O is attached, and FIG. 16C shows the state after the orifice O is attached by being held down with a cover made of a conductive material. The metallic conductive portion R is placed on the bottom surface of the droplet formation unit so as to support the orifice O as shown in FIG. 16. In this case, as shown in FIG. 16A, the conductive portion R has a connection portion R1 at its end that connects to the charging portion 13a. It is important that the conductive portion R itself is completely electrically isolated from ground, and is attached to the droplet formation unit, for example, by a resin screw via an insulating resin block or the like.

[0112] In this embodiment, the orifice O is replaceable and is stacked on the end of the main flow path P13 within the through-hole of the conductive part R, for example, via an O-ring or the like. Then, with the orifice O attached so that it forms a slight protrusion from the surface of the through-hole, it is pressed down with a cover as shown in C of FIG. 16 and fixed with screws or the like. This cover ensures sufficient electrical continuity between the conductive part R and the orifice O, and a charging signal from the charging part 13a is applied to the orifice O via the connection part R1. Note that the method of fixing the replaceable orifice O is not limited to the method using the cover described above, and other methods may be adopted in consideration of user convenience.

[0113] (3-2) Second embodiment of orifice unit U

[0114] The orifice O shown in the first embodiment described above is somewhat difficult to handle during installation and removal, and direct hand contact increases the possibility of contamination. Therefore, in this embodiment, an orifice unit U is formed in which the orifice O is attached to a holder-type conductive part R that supports the orifice O, and the orifice unit U is replaceable, allowing installation and removal from the droplet formation unit. This allows the orifice O and conductive part R to be installed and removed as a single unit, improving user convenience. In this case, by forming part or all of the orifice O and conductive part R from a conductive material, the conductive part R is configured to be electrically conductive with the orifice O, and when the conductive part R is connected to the charging part 13a, the orifice O can be charged.

[0115] The conductive portion R shown in FIG. 17A is made of, for example, metal and has a structure in which a tip-type orifice O is set at its tip. A connection portion R1 for connection to the charging portion 13a is provided on the opposite surface (the outlet side of the liquid column L). A thread is cut into the side as shown in FIG. 17A, allowing for screw-fit attachment to the droplet formation unit as shown in FIG. 17B. The connection position with the charging portion 13a may be omitted from the connection portion R1, and the charging portion 13a may be connected to the droplet formation unit main body. In this case, as in the first embodiment described above, the portion in contact with the orifice unit U may be made of a conductive material, and a charging signal may be connected to it for electrical continuity.

[0116] (3-3) Third embodiment of orifice unit U

[0117] In this embodiment, an orifice unit U is fabricated, including an orifice O and a card-shaped conductive member R supporting the orifice O. The orifice unit U is attached to the end of a flow path through which a particle-containing fluid flows by inserting it laterally into a predetermined gap, similar to a memory card. The card-shaped orifice unit U has an opening (the orifice O) and a groove U1 for attaching an O-ring formed on its outer periphery, as shown in FIG. 18 . Furthermore, a positioning mechanism may be provided, for example, by providing a tapered structure U2 on the end face of the orifice unit U for positioning, so that the orifice O can be accurately positioned relative to the end of the flow path. The orifice unit U of this embodiment may also be replaceable. In this case, a holding member R2 may be provided on the side opposite the insertion direction, allowing the user to hold the unit during replacement.

[0118] In this embodiment, the entire surface of the conductive portion R or a portion of the surface including the orifice O is made of a conductive material and is electrically connected to the connection portion R1, thereby applying a charging signal. As with the second embodiment described above, the connection portion R1 may not be provided, and the charging portion 13a may be connected to the droplet formation unit main body. In this case, as with the first embodiment described above, the portion that comes into contact with the orifice unit U may be made of a conductive material, and the charging signal may be connected to the conductive portion so as to be electrically connected to the conductive portion.

[0119] (3-4) Fourth embodiment of orifice unit U

[0120] In this embodiment, instead of charging the orifice O itself, a conductive portion R is formed at the end of the flow channel through which the particle-containing fluid flows, so that it abuts the orifice O, and charging is performed at the end of the flow channel, i.e., at the inlet of the orifice O. Specifically, as shown in FIG. 19 , a thin-film conductive portion R having a substantially identical opening shape is adhered to the end face of the flow channel so that the sheath fluid directly contacts it. The conductive portion R is then electrically connected to a flow channel holding member or an orifice holding member (orifice holder), and a charging signal is supplied to the flow channel end via the conductive portion R. The conductive portion R can be, for example, a conductive thin-film electrode, which may be formed of metal, or a metal thin film may be formed by vapor deposition, sputtering, plating, or the like so that the electrode is also formed on the side wall near the end of the flow channel.

[0121] In this embodiment, the charging position is about 1 to 2 mm away from the BOP relative to the outlet of the orifice O. However, since the distance from the outlet to the BOP is 10 to 20 mm, this difference is only about 10% of the difference, and therefore the same effect as the above-described embodiments can be obtained. Furthermore, in this embodiment, the orifice O itself does not need to be electrically conductive, and therefore the orifice O can be made of a non-conductor such as resin or ceramic, thereby expanding the options for materials and manufacturing methods. Furthermore, in this case, since the orifice O can be manufactured inexpensively, the orifice O or the orifice holder that holds the orifice O can be disposable. The orifice holder may also have a holder R2 that the user holds when replacing it.

[0122] (4) Chip-type embodiment

[0123] This embodiment is based on the chip system shown in FIG. 4 and A in FIG. In the chip-based orifice system, the orifice O is insulating because it is made of inexpensive resin or the like, assuming disposable use. Therefore, it is necessary to perform a conductive treatment on the orifice O so that it can come into contact with the sheath liquid. In the chips shown in Figures 4 and 20A, the outlet of the orifice O is not located at the end face of the chip, and a hollow portion is formed from the tip of the orifice O to the end face of the chip. Therefore, deposition or sputtering is performed with a mask on the surface or end face of the chip so that a conductive material such as gold, platinum, nickel, or chromium is deposited from the end face of the orifice O to the inner sidewall of the flow channel. In this way, in the case of the chip-based orifice system, the orifice O itself can function as the conductive portion R by making part or all of the orifice O conductive.

[0124] 20B is an enlarged view of the dashed line portion of FIG. 20A. In this embodiment, a thin-wire electrode is provided in the tip loader section of the main body so that a charging signal can be applied to the conductive thin-film-forming portion of the orifice O. When the tip is loaded, the electrode penetrates into the hollow portion of the tip end surface and comes into contact with the conductive thin-film-forming portion. The thin-wire electrode is electrically connected to the charging section 13a, and the sheath liquid is charged via this electrode at the orifice O in the tip. In this embodiment, a position adjustment mechanism may be provided to prevent the thin-wire electrode from coming into contact with the liquid column L ejected from the orifice O within the hollow portion.

[0125] Although the present technology can be applied to the chip type by the above-described method, other configurations are possible without being limited to the above-described embodiment. For example, a method is conceivable in which a metal electrode is inserted near the orifice O by insert molding, a hole is formed on the surface of the chip, and a charging signal is supplied to the metal electrode.

[0126] 4. Second embodiment (particle sorting method)

[0127] The particle sorting method according to this embodiment includes at least an irradiation step, a detection step, and a charging step. Other steps may also be performed as necessary. The specific methods performed in each step are the same as those performed in each section of the particle sorting device 1 according to the first embodiment described above, and therefore will not be described here. [Example]

[0128] The present technology will be described in more detail below based on examples. Note that the examples described below are examples of typical examples of the present technology, and the scope of the present technology should not be construed as being narrow.

[0129] As an example, a particle sorting device with the configuration shown in Figure 8 was used, and a side stream was formed by gradually changing the charging timing using a 10 μsec pulse, which corresponds to one cycle (T) of droplets with a droplet frequency of 100 kHz. In contrast, as a comparative example, we prepared results for the case where a charging signal was connected to the sheath liquid tube attachment part at the position where the sheath liquid was injected into the droplet formation unit as shown in Figure 6, i.e., the conventional charging method A.

[0130] Detailed experimental conditions are described below. ·Droplet frequency: 100kHz Charge signal Pattern: Repeats positive and negative once every 5 droplet cycles *Repeat [+_0_0_0_0_-_0_0_0_0_] Pulse width: T=10μsec at the signal source Pulse voltage: ±160V Deflection plate voltage: ±4.5kV (spacing of entrance straight section: 8mm)

[0131] First, for the charging method according to the present technology (Example) and conventional charging method A (Comparative Example), the probe of an oscilloscope was brought into contact with an aluminum block to which an orifice was attached, and the charging wave shape was observed. FIG. 21 shows the comparison results of the charge signal waveforms between the example and the comparative example.

[0132] 7B, in the comparative example, significant increases in the signal rise time Tr and fall time Tf were observed, and the time Te during which the maximum voltage Vtop was maintained was almost zero. The maximum voltage Vtop was also reduced by about 10% compared to the example.

[0133] Next, for each of the example and comparative example, the phase of the charging pulse was rotated 360° in 10° steps, and the distance between the two side streams split into left and right on the positive and negative sides was measured at a point 170 mm below the top end of the deflection plate. FIG. 22 shows the results of a comparison between the relationship between the side stream deflection distance and the charge signal phase in the example and the comparative example.

[0134] In the example, the charged potential phase showing the maximum deflection distance of 25 mm occupied approximately two-thirds of one period, and in particular, almost no fluctuation was observed in the range of 180° (half period) from phase 150° to 330°. In contrast, in the comparative example, the deflection distance gradually increased with the progression of the charging potential phase, and the phase range in which the maximum deflection distance was maintained was reduced to 130°, from 200° to 330°. In other words, the margin of the charging timing was reduced to about 70% of that of the example. The maximum deflection distance was also reduced by 10% compared to the example.

[0135] The results shown in Fig. 22 can be said to reflect the deterioration of the charging waveform shown in Fig. 21. Therefore, when the charging signal supply point was changed to the orifice closest to the BOP within the droplet formation unit, it was possible to transmit the charging signal output waveform to the tip of the liquid column L with almost no degradation, and it was confirmed that the margin of the charging timing and the deflection angle were improved.

[0136] In the case of conventional charging methods, the degree of signal degradation varies depending on the structure, dimensions, and charging electrode position of the droplet formation unit, and in some cases, even more significant adverse effects than those observed in this experiment may occur. Furthermore, as the droplet frequency increases, the margin for charging timing decreases. In contrast, this technology consistently achieves ideal droplet charging, regardless of the droplet formation unit design.

[0137] This technology also contributes to higher accuracy in actual sorting, even when voltage corrections are made to the charging pulse according to the sorting pattern, and is effective in focusing the sidestream trajectory within the desired range. Specifically, when a droplet is charged, a small amount of charge with reversed polarity is induced in subsequent droplets due to the electrostatic induction phenomenon. For example, if a positive charge of Q is applied to a droplet, a negative charge of 0.2 × Q accumulates in the next droplet, and a negative charge of 0.05 × Q accumulates in the next droplet. This phenomenon is one of the reasons why it is difficult to maintain a constant sidestream trajectory in actual sorting.

[0138] For this reason, defanning, which applies a corrective voltage to the charge signal, is commonly used. In the charge wave form shown in Figure 21, after applying plus or minus I (V), the zero-charged droplet one position behind is given minus or plus 0.1 I (V) instead of the original 0 (V), and the zero-charged droplet two positions behind is given minus or plus 0.025 I (V), thereby correcting the zero-charged droplets to converge correctly to the center. The comparison results of the charge waveforms with and without charge signal correction are shown in Fig. 23. Fig. 23A shows the charge waveform when the charge signal is corrected, and Fig. 23B shows the charge waveform when the charge signal is not corrected.

[0139] In the comparative example, a falling waveform from ±I(V) is superimposed on the signal following the charging pulse, preventing the intended correction from being accurately reflected. Actual sorting requires charging in a variety of random patterns, such as three or more consecutive sorts in the same direction, rather than the repetitive pattern seen in this experiment. Therefore, precise and detailed charge correction is required to ensure consistent convergence of the sidestream trajectory. Therefore, using this technology makes it possible to apply a corrective voltage to the charging signal with near-fidelity, and this effect is particularly pronounced as the droplet frequency increases.

[0140] In addition, the present technology can also employ the following configuration. [1] an irradiation unit that irradiates a part of a flow path through which a fluid containing particles flows with a laser beam; a detection unit that detects light generated by the irradiation of the laser light; an orifice disposed at an end of the flow path and configured to discharge the fluid; a conductive portion disposed near a position where the fluid is turned into droplets; a charging unit that applies an electric charge to the conductive unit based on the optical data detected by the detecting unit; A particle sorting device comprising: [2] The particle sorting device according to [1], wherein a part or all of the orifice is electrically conductive. [3] The particle sorting device according to [2], wherein the conductive portion supports the orifice. [4] The particle sorting device according to [3], wherein the orifice is replaceable. [5] The particle sorting device according to [4], wherein the conductive part is replaceable. [6] The particle sorting device according to [5], wherein the conductive part has a holding part that is held by the user when replacing the conductive part. [7] The particle sorting device according to any one of [2] to [6], wherein the conductive portion includes a connection portion that connects to the charging portion. [8] The particle sorting device according to [1] or [2], wherein the conductive portion is disposed in contact with the orifice. [9] The particle sorting device according to any one of [2] to [7], wherein the orifice is formed in a replaceable tip.

[10] a ground electrode disposed near the location where the fluid is formed into droplets; The particle sorting device according to any one of [1] to [9], wherein the charging unit applies an electric charge to the ground electrode.

[11] The particle sorting device according to any one of [1] to

[10] , wherein the charging unit corrects the charge amount of the droplets.

[12] The particle sorting device according to any one of [1] to

[11] , wherein the conductive portion is arranged downstream in the direction of flow of the fluid from the region irradiated with the laser light.

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

[12] , wherein the conductive portion is formed from one or more conductive materials selected from the group consisting of metals, conductive resins, and non-conductors whose surfaces are made conductive.

[14] The particle sorting device according to any one of [1] to

[13] , wherein the particles are cells.

[15] an orifice, a part of which or the whole of which is electrically conductive; a conductive portion supporting the orifice; An orifice unit for a particle sorting device, comprising:

[16] The orifice unit for a particle sorting apparatus according to

[15] , further comprising a holder that is held by a user when replacing the orifice unit.

[17] The orifice unit for a particle sorting apparatus according to

[15] or

[16] , wherein the conductive portion includes a connection portion connected to a charging portion that imparts an electric charge to the conductive portion.

[18] An orifice unit for a particle sorting device according to any one of

[15] to

[17] , which is attached by a screw-in or side-insertion method to the end of a flow path through which a fluid containing a sheath liquid flows.

[19] An orifice unit for a particle sorting device according to any one of

[15] to

[18] , further comprising a positioning mechanism for attaching to the end of the flow path.

[20] an irradiation step of irradiating a part of a flow path through which a fluid containing particles flows with laser light; a detection step of detecting light generated by the irradiation of the laser light; a charging step of applying an electric charge to a conductive part disposed near a position where the fluid is turned into droplets based on the optical data detected by the detection part; A particle sorting method. [Explanation of symbols]

[0141] 1: Particle separation device 11: Irradiation unit 12: Detection unit 121: Forward scattered light detector 122: Side scattered light detector 13a: Charged part 13b: Deflection plate 13c: Collection container 14: Vibration unit 141: Vibration element 15: Imaging unit 151: Droplet Camera 152: Strobe 16: Break-off control section 17:Analysis Department 18: Storage part 19: Display section 20: User Interface P: Flow path P11: Sample liquid flow path P12: Sheath fluid flow path P13: Main channel P14: Optical detection area D: Droplet BOP: Break-off position O: Orifice R: Conductive part R1: Connection R2: Support part U: Orifice unit for particle sorting device

Claims

1. an irradiation unit that irradiates a part of a flow path through which a fluid containing particles flows with a laser beam; a detection unit that detects light generated by the irradiation of the laser light; an orifice disposed at an end of the flow path and configured to discharge the fluid; a conductive portion disposed near a position where the fluid is turned into droplets; a charging unit that applies an electric charge to the conductive unit based on the optical data detected by the detecting unit; and Some or all of the orifices are electrically conductive; and The conductive portion supports the orifice.

2. The particle sorting device of claim 1 , wherein the orifice is replaceable.

3. The particle sorting device according to claim 2 , wherein the conductive portion is replaceable.

4. The particle sorting device according to claim 3 , wherein the conductive portion has a holder that is held by a user when the conductive portion is replaced.

5. The particle sorting device according to claim 1 , wherein the conductive portion includes a connection portion that connects to the charging portion.

6. The particle sorting device according to claim 1 , wherein the conductive portion is disposed in contact with the orifice.

7. The particle sorting device of claim 1 , wherein the orifice is formed in a replaceable tip.

8. a ground electrode disposed near the location where the fluid is formed into droplets; The particle sorting apparatus according to claim 1 , wherein the charging unit applies a charge to the ground electrode.

9. The particle sorting device according to claim 1 , wherein the charge unit corrects the amount of charge on the droplets.

10. The particle sorting device according to claim 1 , wherein the conductive portion is disposed downstream in a flow direction of the fluid from a region irradiated with the laser light.

11. 2. The particle sorting device according to claim 1, wherein the conductive portion is formed from one or more conductive materials selected from the group consisting of metals, conductive resins, and non-conductors whose surfaces are made conductive.

12. The particle sorting device according to claim 1 , wherein the particles are cells.

13. an orifice, a part of which or the whole of which is electrically conductive; a conductive portion supporting the orifice; An orifice unit for a particle sorting device, comprising:

14. The orifice unit for a particle sorting apparatus according to claim 13 , further comprising a holder that is held by a user when replacing the orifice unit.

15. The orifice unit for a particle sorting apparatus according to claim 13 , wherein the conductive portion includes a connection portion connected to a charging portion that applies an electric charge to the conductive portion.

16. 14. The orifice unit for a particle sorting apparatus according to claim 13, which is attached by a screw-in method or a side-insertion method to an end of a flow path through which a fluid containing particles flows.

17. The orifice unit for a particle sorting apparatus according to claim 16, further comprising a positioning mechanism for attaching the orifice unit to an end of the flow channel.

18. an irradiation step of irradiating a part of a flow path through which a fluid containing particles flows with laser light; a detection step of detecting light generated by the irradiation of the laser light; a charging step of applying an electric charge to a conductive part disposed near a position where the fluid is formed into droplets based on the optical data detected in the detecting step; and an orifice for discharging the fluid is disposed at the end of the flow path; Some or all of the orifices are electrically conductive; and The particle sorting method, wherein the conductive portion supports the orifice.

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