JP6706011B2 - Particle sorting device, particle sorting method and program - Google Patents

Description

The present technology relates to a particle sorting device, a particle sorting method, and a program. More specifically, it relates to a technique for separating and collecting particles based on the result of analysis by an optical method or the like.

BACKGROUND ART Conventionally, an optical measurement method using flow cytometry (flow cytometer) has been used for analysis of cells, microorganisms, and biologically related microparticles such as liposomes. A flow cytometer is a device that irradiates light to the microparticles that flow in the flow path formed in a flow cell or microchip, and detects the fluorescence or scattered light emitted from each microparticle and analyzes it. is there.

Some flow cytometers have a function of separating and collecting only microparticles having specific characteristics based on the analysis results. In particular, a microparticle device for sorting cells is called a "cell sorter". Has been. In a cell sorter, generally, a fluid is discharged from its flow path into droplets by vibrating a flow cell or a microchip with a vibrating element or the like (see Patent Documents 1 and 2).

The droplets separated from the fluid are charged with plus (+) or minus (-) charges, and then the traveling direction of the droplets is changed by a deflecting plate or the like, and the droplets are collected in a predetermined container or the like. Also, conventionally, a technique has been proposed in which specific cells are distributed one by one to each reaction site of a base material used in a PCR (Polymerase Chain Reaction) method or the like by utilizing the sorting function of the cell sorter. (See Patent Document 3).

Japanese Patent Publication No. 2007-532874 JP, 2010-190680, A Japanese Patent Publication No. 2010-510782

However, in a conventional particle sorter such as a cell sorter, when particles of different sizes are mixed in the sample liquid, the advancing direction of the droplet becomes unstable, and the droplet is not distributed to a predetermined container or reaction site. There is a problem that the collection accuracy and the separation efficiency decrease. For this reason, in the conventional particle sorting apparatus, the size of particles that can be stably collected is set to 1/5 or less of the orifice diameter. In that case, the orifice is adjusted according to the size of the particles to be sorted. It is necessary to increase the diameter, which causes a problem that the preparative rate is reduced.

Therefore, it is a main object of the present disclosure to provide a particle sorting apparatus, a particle sorting method, and a program capable of accurately sorting even if the particles to be sorted are large.

The present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, if large cells or particles are present in the droplet, the fluid discharged from the orifice becomes a break-off (Break- It has been found that the timing of off) tends to be delayed. If the break off timing is misaligned, droplets containing large cells will fall inwardly of the properly charged droplets because the droplets will not receive the proper charge. That is, if the particles to be sorted include large particles, the break-off timing becomes unstable, and as a result, the angle of the side stream becomes unstable, and the droplets to be sorted are splashed. I found it to occur.

Therefore, in the present disclosure, the charge application end time is adjusted according to the break-off timing delay that occurs when sorting large particles. As a result, it is possible to stably apply an electric charge even to a liquid droplet containing a large size particle and reduce the occurrence of splashing.

That is, the particle henchmen winder engaged Ru in the present disclosure includes a charge section that applies an electric charge to at least a portion of the liquid droplets ejected from the orifice to generate a fluid stream, the size of the particles contained in the droplets And a charge control unit that adjusts the charge application end time in the charging unit based on the corresponding mode.
The charge control unit may change the timing of charge application according to the size of the particles contained in the droplet, and may change the charge application time according to the size of the particles contained in the droplet. You may.
Furthermore, the particle sorting apparatus according to the present disclosure has a forward scattered light detection unit that irradiates particles flowing through the flow path with light and detects forward scattered light generated from the particles by the light irradiation. The charge control unit may adjust the charge application end time based on the detection result of the forward scattered light detection unit. In this case, when the intensity of the forward scattered light detected by the forward scattered light detection unit is equal to or higher than a preset threshold, the charge control unit imparts a charge more than when the intensity of the forward scattered light is less than the threshold. It is possible to control the charging section so that the timing of is delayed. In addition, the forward scattered light detection unit has a delay amount calculation unit that calculates the delay amount of the charge application timing based on the intensity of the forward scattered light, and the charge control unit is calculated by the delay amount calculation unit. It is also possible to control the charging unit so that the timing of charge application is delayed by the amount of the delay amount. Furthermore, the charge control unit, when the intensity of the forward scattered light detected by the forward scattered light detection unit is equal to or greater than a preset threshold value, the charge application time is longer than when the intensity of the forward scattered light is less than the threshold value. It is also possible to control the charging section so that the charging time becomes longer. In addition, it has an application time calculation unit that calculates a charge application time based on the intensity of the forward scattered light detected by the forward scattered light detection unit, and the charge control unit is calculated by the application time calculation unit. It is also possible to control the charging unit so that the charge is given for the time.
In addition, the orifice is formed in a replaceable microchip, and the charging section is arranged in contact with a sheath liquid and/or a sample liquid flowing in a flow path provided in the microchip. An electrode may be provided and the orifice may be formed in the flow cell.
Further, the mode may be composed of two modes, a normal mode and a large particle mode. In this case, in the normal mode and the large particle mode, the timing of charge application and the charge application time may be set in advance.

Engaging Ru particle henchmen preparative method of the present disclosure includes a step of applying an electric charge to at least a portion of the liquid droplets ejected from the orifice to generate a fluid stream, responsive to the size of the particles contained in the droplets The charge application end time is adjusted based on the mode.

A program according to the present disclosure has a function of adjusting a charge application end time based on a mode according to a size of a particle included in a droplet discharged from an orifice that generates a fluid stream. To run.

According to the present disclosure, even if the particles to be sorted are large, they can be sorted accurately.
Note that the effects described here are not necessarily limited, and may be any effects described in the present disclosure.

It is a figure which shows typically the structural example of the particle sorting apparatus of 1st Embodiment of this indication. It is a flowchart figure which shows the charge provision end time adjustment method by changing charge provision timing. It is a flowchart figure which shows the charge provision end time adjustment method by changing charge provision time. It is a figure which shows the relationship of charge provision and a droplet formation state in a "normal mode." FIG. 6 is a diagram showing a droplet formation state when the charge application end time is adjusted by changing the charge application timing. FIG. 6 is a diagram showing a droplet formation state when the charge application end time is adjusted by changing the charge application time. It is a block diagram which shows the structural example of the charge control mechanism of the particle sorting apparatus of the 1st modification of 1st Embodiment of this indication. It is a flowchart figure which shows the charge provision completion time adjustment method in the particle sorting apparatus of the 1st modification of 1st Embodiment of this indication. It is a block diagram showing an example of composition of a charge control mechanism of a particle sorting device of the 2nd modification of a 1st embodiment of this indication. It is a flowchart figure which shows the charge provision completion time adjustment method in the particle sorting apparatus of the 2nd modification of 1st Embodiment of this indication. It is a figure which shows typically the structural example of the particle sorting apparatus which concerns on 2nd Embodiment of this indication. It is a figure which shows typically the example of the image imaged by the camera 12 shown in FIG. It is a figure which shows typically the structural example of the particle fractionation apparatus of the 3rd Embodiment of this indication. 13A is a diagram schematically showing the relationship between a side stream and a well plate in the particle sorting apparatus shown in FIG. 13, and B is a schematic diagram showing the relationship between a side stream and a well plate in a conventional particle sorting apparatus. It is a figure. It is a side view which shows typically the state when a well plate is mounted in the plate holder in which the plate mounting part inclines. It is a flowchart figure which shows the operation example of the particle fractionation apparatus shown in FIG.

Hereinafter, modes for carrying out the present disclosure will be described in detail with reference to the accompanying drawings. Note that the present disclosure is not limited to the embodiments described below. The description will be given in the following order.

1. First embodiment (an example of a particle sorting apparatus that adjusts the end time of charge application according to the size of particles)
2. First Modification of First Embodiment (Example of Particle Sorting Apparatus Having Delay Amount Calculation Unit)
3. Second Modified Example of First Embodiment (Example of Particle Sorting Apparatus Equipped with Addition Time Calculation Unit)
4. Second Embodiment (Example of Particle Sorting Apparatus Controlling Vibration Element Based on Imaged Droplet Image Together with Adjustment of Charge Application End Time)
5. Third Embodiment (Example of Particle Sorting Device in which Droplet Collection Plate is Obliquely Arranged)

<1. First Embodiment>
First, the particle sorting apparatus according to the first embodiment of the present disclosure will be described. FIG. 1 is a diagram showing a schematic configuration of a particle sorting apparatus according to a first embodiment of the present disclosure.

[Overall configuration of device]
The particle sorting apparatus 1 of the present embodiment separates and collects particles based on the result of analysis by an optical method or the like, and as shown in FIG. 1, the microchip 2, the vibration element 3, and the charging unit 4 are arranged. , A charge controller 7, deflection plates 5a, 5b, and the like.

[Particle]
The particles analyzed and fractionated by the particle fractionation apparatus 1 of the present embodiment include biologically related fine particles such as cells, microorganisms and ribosomes, or synthetic particles such as latex particles, gel particles and industrial particles. included.

The living body-related microparticles include chromosomes, ribosomes, mitochondria, organelles (organelles), etc. that make up various cells. In addition, the cells include plant cells, animal cells, blood cells, and the like. Further, the microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, fungi such as yeast. The bio-related microparticles may include bio-related macromolecules such as nucleic acids, proteins and complexes thereof.

On the other hand, examples of the industrial particles include particles formed of an organic polymer material, an inorganic material, a metal material, or the like. As the organic polymer material, polystyrene, styrene/divinylbenzene, polymethylmethacrylate, etc. can be used. Further, as the inorganic material, glass, silica, magnetic material or the like can be used. As the metal material, for example, gold colloid and aluminum can be used. The shape of these particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

[Microchip 2]
The microchip 2 includes a sample inlet 22 into which a liquid (sample liquid) containing particles to be sorted is introduced, a sheath inlet 23 into which a sheath liquid is introduced, and a suction outlet 24 for eliminating clogging and air bubbles. Has been formed. In this microchip 2, the sample liquid is introduced into the sample inlet 22, merges with the sheath liquid introduced into the sheath inlet 23, and is sent to the sample channel, and the orifice 21 provided at the end of the sample channel. Is discharged from.

Further, a suction flow path communicating with the suction outlet 24 is connected to the sample flow path. This suction channel is for eliminating clogging and air bubbles by causing negative pressure in the sample channel to temporarily reverse the flow when clogging or air bubbles occur in the sample channel. A negative pressure source such as a vacuum pump is connected to 24.

The microchip 2 can be formed of glass or various plastics (PP, PC, COP, PDMS, etc.). It is desirable that the material of the microchip 1 is transparent to the measurement light emitted from the photodetector described later, has a small amount of autofluorescence, and has a small wavelength dispersion, and thus has a small optical error.

The molding of the microchip 2 can be performed by wet etching or dry etching of a glass substrate, or nanoimprinting, injection molding, or machining of a plastic substrate. The microchip 2 can be formed, for example, by sealing a substrate on which a sample channel or the like is molded with a substrate of the same material or a different material.

[Vibration element 3]
The vibrating element 3 is for generating a fluid stream (flow of droplets) S by applying minute vibrations to the liquid flowing in the flow path to turn the fluid discharged from the orifice 21 into droplets. , A piezoelectric element or the like can be used. The vibrating element 3 may be provided at a position where vibration can be applied to the liquid flowing in the flow path, and in addition to being disposed inside the microchip 2 or in contact with the microchip 2, the vibrating element 3 can be used for the flow of a sheath pipe or the like. It may be attached to a pipe for introducing the liquid into the passage.

[Charging part 4]
The charging unit 4 imparts positive or negative charges to the liquid droplets discharged from the orifice 21, and the charge electrode 41 and a voltage source (voltage supply unit 42) that applies a predetermined voltage to the electrode 41. Etc. The charging electrode 41 is disposed in contact with the sheath liquid and/or the sample liquid flowing in the flow path, and imparts an electric charge to the sheath liquid and/or the sample liquid. For example, the charging electrode inlet of the microchip 2 is used. Inserted in.

In FIG. 1, the charging electrode 41 is arranged so as to come into contact with the sample liquid, but the present disclosure is not limited to this, and it may be arranged so as to come into contact with the sheath liquid. You may arrange|position so that it may contact both a liquid and a sheath liquid. However, in consideration of the influence on the cells to be sorted, it is desirable that the charging electrode 41 is arranged so as to be in contact with the sheath liquid.

In this way, by charging a desired droplet by positively or negatively charging it, it becomes possible to separate droplets containing arbitrary particles by an electric force. Further, by synchronizing the timing of charging by the charging unit 4 and the voltage supplied to the vibration element 3, it becomes possible to charge only an arbitrary droplet.

[Deflection plates 5a, 5b]
The deflecting plates 5a and 5b change the advancing direction of each droplet in the fluid stream S by the electric force acting between the deflecting plates 5a and 5b and the electric charge applied to the droplet, and guide the droplets to the predetermined recovery containers 6a to 6c. They are arranged opposite to each other with the fluid stream S in between. For the deflection plates 5a and 5b, for example, commonly used electrodes can be used.

Different positive or negative voltages are applied to the deflecting plates 5a and 5b, and when the charged droplets pass through the electric field formed by these, an electric force (Coulomb force) is generated and each liquid is generated. The drops are attracted in the direction of either deflector 5a, 5b. In the particle sorting apparatus 1, the direction of the flow (side stream) of the liquid droplets attracted by the electric field can be controlled by changing the positive/negative charge of the liquid droplets and the amount of charge, and thus a plurality of mutually different liquid droplets can be controlled. It is possible to sort particles at the same time.

[Recovery containers 6a to 6c]
The collection containers 6a to 6c are for collecting the liquid droplets that have passed between the deflection plates 5a and 5b, and a general-purpose plastic tube, glass tube, or the like can be used for experiments. It is preferable that these recovery containers 6a to 6c are replaceably arranged in the apparatus. Further, among the collection containers 6a to 6c, those for receiving particles not to be sorted may be connected to a drainage path of the collected droplets.

The number and types of recovery containers arranged in the particle sorting apparatus 1 are not particularly limited. For example, when arranging more than three recovery containers, each droplet is guided to any one of the recovery containers depending on the presence or absence of the electric action force between the deflection plates 5a and 5b and the magnitude thereof. , Should be collected. Further, instead of the recovery containers 6a to 6c, a base material in which a plurality of reaction sites (wells) are formed can be used and one specific particle can be distributed to each reaction site.

[Charge control unit 7]
The charge control unit 7 adjusts the charge application end time in the charging unit 4 according to the size of the particles contained in the droplet. The method for determining the particle size is not particularly limited, but it can be determined based on, for example, the detection result of the forward scattered light measured by the photodetector described later. In that case, the charge control unit 7 determines, for example, the time to start charge application (charge application timing) or the charge based on whether or not the intensity of the forward scattered light detected by the light detection unit is a specific value (threshold value) or more. The applied time (charge application time) is changed to adjust the charge application end time.

Specifically, when the intensity of the forward scattered light is equal to or higher than a preset threshold value, the charge control unit 7 delays the charge application timing as compared with the case where the intensity of the forward scattered light is less than the threshold value. Alternatively, the charging unit 4 may be controlled so that the charge application time becomes longer. As a result, even if the particles to be sorted are large, the charge can be applied at an appropriate timing, so that the droplets can be stably guided by the polarizing plates 5a and 5b.

[Light detector]
Further, in the particle sorting apparatus 1 of the present embodiment, for example, light (excitation light) is applied to a predetermined portion of the sample channel, and light (measurement target light) generated from particles flowing through the sample channel is detected. An optical detection unit (not shown) is provided. The photodetector can be configured similarly to conventional flow cytometry. Specifically, a laser light source, an irradiation system that includes a condenser lens that collects and irradiates particles with laser light, a dichroic mirror, a bandpass filter, and the light to be measured that is emitted from particles by irradiation with laser light. And a detection system for detecting.

The detection system is composed of, for example, a PMT (Photo Multiplier Tube) or an area image pickup device such as a CCD or a CMOS device. The irradiation system and the detection system may be configured by the same optical path or may be configured by separate optical paths. Further, the measurement target light detected by the detection system of the light detection unit is light generated from particles by irradiation of excitation light, and for example, various kinds of forward scattered light, side scattered light, Rayleigh scattering, Mie scattering, etc. It can be scattered light, fluorescence, or the like.

Among these measurement target lights, the forward scattered light changes in intensity in proportion to the surface area of cells and serves as an index for evaluating the size of particles. Therefore, the particle sorting apparatus 1 of the present embodiment preferably includes a forward scattered light detection unit that detects forward scattered light, which facilitates adjustment of the charge application end time by the charge control unit 7. It becomes possible to do it.

[Other]
In addition to the above-mentioned parts, the particle fractionation device 1 of the present embodiment supplies stable air pressure to the sheath liquid and the sample liquid, and therefore, a pneumatic pressure device such as a compressor, a pressure sensor, or the like. The air pressure detector may be provided. Thereby, the sheath flow and the sample flow can be stably formed, and stable droplet formation can be realized.

[motion]
Next, regarding the operation of the particle sorting apparatus 1 of the present embodiment, that is, the method of sorting particles using the particle sorting apparatus 1, the case where the charge amount is adjusted using the detection result of the forward scattered light will be described. An example will be described.

When the particles are sorted by the particle sorting apparatus 1 of the present embodiment, the sample liquid containing the particles to be sorted is introduced into the sample inlet 22 and the sheath liquid is introduced into the sheath inlet 23. Then, for example, the photodetection unit detects the optical characteristics of the particles, and at the same time, detects the flow velocity (flow velocity) of the particles and the distance between the particles. The detected optical characteristics, flow velocity, interval, etc. of the particles are converted into an electrical signal and output to the overall control unit (not shown) of the device.

The laminar flow of the sample liquid and the sheath liquid that has passed through the light irradiation portion of the sample flow path is discharged from the orifice 21 to the space outside the microchip 2. At this time, the vibrating element 3 vibrates the liquid such as the sheath liquid flowing through the flow path to make the fluid discharged from the orifice 21 into droplets. Then, each droplet is changed in its traveling direction by the deflecting plates 5a and 5b based on the detection result of the light detecting section, guided to the predetermined collecting containers 6a to 6c, and collected.

At this time, in the particle sorting apparatus 1 of the present embodiment, the charge application end time in the charging unit 4 is adjusted according to the size of the particles contained in the droplet. FIG. 2 is a flow chart showing a method of adjusting the charge application end time by changing the charge application timing, and FIG. 3 is a flow chart showing the method of adjusting the charge application end time by changing the charge application time. Further, FIG. 4 is a diagram showing the relationship between the charge application and the droplet formation state in the “normal mode”. Further, FIG. 5 is a diagram showing a droplet forming state when the charge application end time is adjusted by changing the charge application timing, and FIG. 6 is a view showing a droplet forming state when the charge application end time is adjusted by changing the charge application time.

The charge control unit 7 can adjust the charge application end time for the droplet containing each particle based on the intensity Sfsc of the forward scattered light, for example. Specifically, for particles whose forward scattered light intensity is equal to or greater than a preset threshold value, the charge application end time can be automatically adjusted by controlling, for example, the charge application timing or the charge application time. ..

For example, when the charge application end time is adjusted by changing the charge application timing, each particle flowing in the flow path is detected and its forward scattered light intensity (Sfsc) is determined as shown in FIG. get. Then, when the forward scattered light intensity of the particle is less than the threshold value (Tfsc), charges are applied at the normal timing shown in FIG. 4, and when the forward scattered light intensity is equal to or higher than the threshold value (Tfsc), shown in FIG. The charges are applied at a timing delayed from the normal timing.

That is, when the intensity (Sfsc) of the forward scattered light is equal to or higher than the threshold value (Tfsc) set in advance, the charge control unit 7 is more charged than when the intensity (Sfsc) of the forward scattered light is less than the threshold value (Tfsc). The charging unit 4 is controlled so that the application timing is delayed. Here, the delay amount of the charge application timing can be appropriately selected based on the assumed particle size and the like, and may be set in advance.

Also, for example, when the charge application end time is adjusted by changing the charge application end time, as shown in FIG. 3, each particle flowing in the flow channel is detected and its forward scattered light intensity ( Sfsc) is acquired. Then, when the forward scattered light intensity of the particle is less than the threshold value (Tfsc), charges are applied with the normal length shown in FIG. 4, and when the forward scattered light intensity is equal to or more than the threshold value (Tfsc), The charge is applied for a longer time than the normal shown.

That is, when the intensity (Sfsc) of the forward scattered light is equal to or higher than the threshold value (Tfsc) set in advance, the charge control unit 7 is more charged than when the intensity (Sfsc) of the forward scattered light is less than the threshold value (Tfsc). The charging unit 4 is controlled so that the application time becomes longer. Here, the extension time of the charge application can be appropriately selected based on the assumed particle size and the like, and may be set in advance.

As described above, by delaying the charge application timing or increasing the charge application time for a droplet containing a large particle, it is possible to reliably charge the droplet even if the break-off timing is delayed. Can be given. The "normal charge application timing" and "normal charge application time" described above adjust the amplitude (drive value) supplied to the vibrating element 3 before measurement so that the most stable side stream can be obtained. It is decided at the time. At this time, the relationship between the charge waveform and the droplet formation is the timing at which the charge application ends immediately after the droplet breaks off, and this is referred to as the “normal mode”.

On the other hand, in order to stabilize the sorting of large-sized particles, it is important to apply the electric charge immediately after the droplet breaks off. Comparing the two charge application end time adjustment methods described above, the method of increasing the charge application time is to increase the charge application time with a margin so that the liquid charge can be applied regardless of break-off timing fluctuations. An electric charge can be surely given to the droplet. However, in this method, when the break-off timing varies widely, the amount of charge applied to each droplet may vary.

On the other hand, in the method of delaying the charge application timing, the charge application time is not changed and the total charge amount applied to each droplet is constant, so that the above-mentioned problem of charge amount variation does not occur. In the method of delaying the charge application timing, if the delay amount of break-off timing can be accurately estimated by the method described later, the charge application timing is delayed accordingly, and the charge amount of each droplet is delayed. It becomes possible to stabilize the collection while keeping the value constant.

Furthermore, the adjustment of the charge application end time described above is performed by creating a program for realizing the function of changing the charge application timing and the charge application time according to the size of the particles contained in the droplet, and the particle sorting apparatus It can be automatically implemented by mounting it in the first charge control unit 7. Alternatively, the configuration may be such that the user selects and executes the “normal mode” and the “large diameter particle mode” as necessary.

As described above in detail, in the particle sorting apparatus of the present embodiment, since the adjustment of the charge application end time in the charging unit is adjusted according to the size of the particles contained in the droplet, it is possible to remove the particles having a large size. The electric charges can be stably applied to the contained liquid droplets. As a result, even if the particles to be sorted are large, it is possible to reduce the disturbance of the side stream due to the delay of the break off timing and to sort the particles with high accuracy.

As a result, in the conventional fractionation device, when the large particles were fractionated, the orifice diameter had to be increased, but according to the present disclosure, even if the particles are large, the orifice diameter should be increased. Since it is not necessary to perform this, it is possible to perform the sorting at a higher speed than in the past.

In the above-described first embodiment, the case where the microchip 2 is used has been described as an example, but the present disclosure is not limited to this, and the same applies when a flow cell is used instead of the microchip 2. The effect is obtained.

<2. First Modified Example of First Embodiment>
Next, a particle sorting apparatus according to a first modified example of the first embodiment of the present disclosure will be described. FIG. 7 is a block diagram showing a configuration example of a charge control mechanism of the particle sorting apparatus of the present modified example, and FIG. 8 is a flow chart diagram showing the charge application end time adjusting method.

[Device configuration]
The forward scattered light intensity Sfsc has a value that is substantially proportional to the surface area (size) of the particle. Therefore, by setting the delay amount of the charge application timing on the basis of the forward scattered light intensity Sfsc of each particle, the fractionation is stabilized. The property can be further improved. Therefore, as shown in FIG. 7, the particle sorting apparatus of the present modified example calculates the delay amount D of the charge application timing on the basis of the intensity Sfsc of the forward scattered light detected by the photodetector 8, the delay amount calculation unit. 9 is provided.

[motion]
In the particle sorting apparatus of this modified example, the charge control unit 7 controls the charging unit 4 so that the charge application timing is delayed by the delay amount D calculated by the delay amount calculation unit 9. Specifically, as shown in FIG. 8, first, the light detection unit 8 detects particles and acquires the forward scattered light intensity Sfsc thereof. Then, based on the forward scattered light intensity Sfsc data, the delay amount calculation unit 9 calculates the delay amount D of the charge application timing. The data of the delay amount D is sent to the charging control unit 7 and used for controlling the charge application by the charging unit 4.

As described above, in the particle sorting apparatus of the present modification, the delay amount D of the charge application timing is calculated from the intensity Sfsc of the forward scattered light, and the charge control unit 7 determines the charge application timing of the charge unit 4 based on the calculated value. Since it is controlled, the preparative stability can be further improved.

The configuration and effects of the particle sorting apparatus of this modification other than those described above are the same as those of the first embodiment described above.

<3. Second Modified Example of First Embodiment>
Next, a particle sorting apparatus according to a second modified example of the first embodiment of the present disclosure will be described. FIG. 9 is a block diagram showing a configuration example of a charge control mechanism of the particle sorting apparatus of the present modified example, and FIG. 10 is a flow chart diagram showing the charge application end time adjusting method.

[Device configuration]
As described above, the forward scattered light intensity Sfsc has a value that is substantially proportional to the surface area (size) of the particle. Therefore, by setting the charge application time based on the forward scattered light intensity Sfsc of each particle, fractionation is performed. The stability of can be further improved. Therefore, in the particle sorting apparatus of the present modified example, as shown in FIG. 9, an application time calculation unit 10 that calculates the charge application time T based on the intensity Sfsc of the forward scattered light detected by the light detection unit 8 is provided. ing.

[motion]
In this particle sorting apparatus, the charging control unit 7 controls the charging unit 4 so that the droplets are charged during the time T calculated by the applying time calculation unit 10. Specifically, as shown in FIG. 10, first, the light detection unit 8 detects particles and acquires the forward scattered light intensity Sfsc thereof. Then, based on the forward scattered light intensity Sfsc data, the charge application time T is calculated by the application time calculation unit 10. The data of the charge application time T is sent to the charge control unit 7 and used for controlling the charge application by the charging unit 4.

As in the particle sorting apparatus of this modification, the charge application time T is calculated from the intensity Sfsc of the forward scattered light, and the charge control unit 7 controls the charge application time by the charging unit 4 based on the calculated value. Thus, the preparative stability can be further improved.

The configuration and effects of the particle sorting apparatus of this modification other than those described above are the same as those of the first embodiment described above.

<4. Second Embodiment>
Next, a particle sorting apparatus according to the second embodiment of the present disclosure will be described. FIG. 11: is a figure which shows typically the structural example of the particle sorting apparatus which concerns on 2nd Embodiment of this indication. As shown in FIG. 11, in addition to the configuration of the first embodiment described above, the particle sorting apparatus 11 of the present embodiment has an imaging element (camera) 12 that acquires an image of a fluid or a droplet, and a camera 12. The vibration control unit 14 that controls the drive voltage of the vibrating element 3 based on the image captured in 1. is provided.

[Image sensor (camera) 12]
The imaging element (camera) 12 is a fluid and droplets before being formed into droplets at a position (break off point BP) where the laminar flow of the sample liquid and the sheath liquid discharged from the orifice 21 is formed into droplets. Is to be imaged. In addition to the image pickup device such as a CCD or a CMOS camera, various image pickup devices such as a photoelectric conversion device can be used to image the fluid and the liquid droplets.

Further, the camera 12 is preferably provided with a position adjusting mechanism 15 for changing the position thereof. As a result, the position of the camera 12 can be easily controlled by an instruction from the vibration control unit 14 described later. In addition, the particle sorting apparatus 11 of the present embodiment may be provided with a light source (not shown) that illuminates an imaging region together with the camera 12.

[Voltage supply unit 13]
The voltage supply unit 13 supplies a drive voltage to the vibration element 3. The drive voltage of the vibrating element 3 is supplied according to a sine wave in order to form a stable droplet, and is controlled by two of a frequency (clock value) and an amplitude (drive value).

[Excitation controller 14]
The vibration control unit 14 controls the drive power of the vibration element 3 based on the image captured by the camera 12, and also controls the position of the camera 12 as necessary. Specifically, the state of the fluid in the image before being formed into droplets, or the state of the satellite droplets existing between the break off point and the droplet closest to the break off point, or The voltage supply unit 13 and the position adjusting mechanism 15 are controlled based on both of them.

The vibration control unit 14 can be configured with an information processing device including, for example, a general-purpose processor, a main storage device, and an auxiliary storage device. In that case, the image data captured by the image sensor such as the camera 12 is input to the vibration control unit 14 and the programmed control algorithm is executed to automatically control the voltage supply unit 13 and the position adjustment mechanism 15. It becomes possible. Such a computer program may be stored in a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, or may be distributed via a network.

[motion]
Next, the operation of the particle sorting apparatus 11 of this embodiment will be described. In addition to the control of the charging section 4 by the charging control section 7, the particle sorting apparatus 11 of the present embodiment acquires images of fluid and droplets at the break-off point with the camera 12, and based on the images, The vibration control unit 14 controls the vibration element 3.

(Acquire droplet image)
The method of imaging the fluid and the liquid droplets by the imaging device (camera) 12 is not particularly limited, but, for example, by causing the light source to emit light for a certain period of time in each liquid droplet formation cycle, the liquid at a specific timing of liquid droplet formation. A drop image can be acquired. It is also possible to confirm how droplets are formed in one cycle by changing the light source emission timing in the droplet formation clock. The droplet forming frequency is about 10 kHz to 30 kHz, and the image pickup element (camera) 12 is usually about 30 fps. Therefore, one droplet image is formed by superimposing hundreds to thousands of droplets. Become.

(Control of drive voltage)
When the drive voltage of the vibrating element 3 is controlled by the vibration control unit 14, for example, an image (reference image) obtained by previously adjusting the fluid or liquid droplets to an optimum state is prepared, and the image at the time of sorting is displayed. Adjust the drive voltage to match the reference image. FIG. 12 is a diagram schematically showing an example of an image captured by the camera 12. The comparison between the reference image and the image at the time of sorting is performed by comparing the distance from the break-off point BP to the first satellite SD1 (upper space of the first satellite) d, the width of the constricted portion in the fluid immediately before being made into droplets ( It can be performed by the liquid column constriction width) w or the like.

The distance d between the upper portions of the first satellite, the width w of the liquid column, and the length L of the liquid column (the position of the break off point BP) are closely related to each other, and the length L of the liquid column and the upper portion of the first satellite The interval d and the liquid column neck width w are indices that directly indicate the stability of the break off point BP. The droplet shape of the fluid stream S can be stabilized by controlling the drive voltage of the vibrating element 3 based on the values of the first satellite upper space d and the liquid column neck width w.

For example, the vibration control unit 14 drives the vibrating element 3 so that the first satellite upper space d in the image during sorting becomes the same as the first satellite upper space dref in the reference image 71 shown in FIG. Control the voltage. When the drive voltage of the vibrating element 3 is increased, the value of the first satellite upper space d is increased, and conversely, when the drive voltage of the vibrating element 3 is decreased, the value of the first satellite upper space d is decreased. 14 can control the drive voltage of the vibration element 3 by utilizing this relationship.

The first satellite top spacing d is sensitive to changes in the droplet shape of the fluid stream S. Therefore, by continuously adjusting the first satellite upper space d so as to match the first satellite upper space dref of the reference image 71, the droplet shape at the time of sorting is maintained in the same stable state as the reference image. It becomes possible to do.

Further, the drive voltage of the vibration element 3 can be controlled by using the liquid column constriction width w instead of the above-mentioned first satellite upper portion distance dref. In that case, the drive voltage of the vibrating element 3 is controlled so that the value of the liquid column neck width w in the image during fractionation becomes the same as the liquid column neck width wref in the reference image 71 shown in FIG. The value of the liquid column neck width w decreases when the drive voltage of the vibrating element 3 is increased, and the value of the liquid column neck width w increases when the driving voltage of the vibrating element 3 is decreased. Can be used to control the drive voltage of the vibration element 3.

The liquid column constriction width w also changes sensitively in response to the change in the droplet shape of the fluid stream S, similarly to the above-mentioned first satellite upper space dref. Therefore, by continuing to adjust the liquid column waist width w so as to match the liquid column waist width wref of the reference image 71, the fluid stream S can be maintained in a stable state, and the break off point BP can be maintained. Position is stable.

It should be noted that the drive voltage control of the vibration element 3 by the vibration control unit 14 can use either one of the first satellite upper space d and the liquid column neck width w as an index, but use both of these as indices. Thereby, the droplet shape in the fluid stream S can be further stabilized. Alternatively, the driving voltage of the vibration element 3 can be controlled based on only the fluid state without using the satellite droplet state.

(Control of camera position)
When the sheath liquid temperature fluctuates according to the change of the environmental temperature during the fractionation, the droplet interval in the fluid stream S changes due to the fluctuation of the flow velocity due to the change of the viscosity, and the position of the break off point BP, that is, the liquid column. The length L varies. As a result, the number of liquid droplets FD in the liquid column in the image may change, and the break-off point BP may not be stably detected and discriminated.

Therefore, in the particle sorting apparatus 11 of the present embodiment, the position of the camera 12 can be moved by the vibration control unit 14 according to the change of the liquid column length L in the image, if necessary. In this way, when the position of the camera 12 is made to follow the position change of the break off point BP, the value of the liquid column length L in the image can be kept constant. As a result, in the preparative image, the break-off point BP is stably held at the predetermined position corresponding to the reference image, so that the number of droplets FD in the liquid column is kept constant and preliminarily adjusted. It is possible to maintain the drop delay time for a long time.

As a method of holding the position of the break-off point BP in the image constant, there is a method of changing the cut-out position of the image in addition to the method of moving the camera 12 itself. For example, a wide-angle camera is used to capture an image of fluid and droplets, and an image including the break-off point BP is cut out from the image and used for control by the vibration control unit 14. In this case, when the position of the break-off point BP changes, the image cutout position is changed so as to suppress the change in the value of the liquid column length L. As a result, it is possible to realize the control of the imaging position, which is accompanied by the movement of the break-off point BP.

Since the particle sorting apparatus of the present embodiment controls the drive voltage of the vibration element based on the state of the fluid stream S in addition to adjusting the charge application end time, the break off point BP can be set with high accuracy. Can be maintained. This stabilizes not only the charging of the droplets but also the formation of the droplets. Therefore, even when the particles to be sorted are large, it is possible to sort them at high speed and with high accuracy.

The configuration and effects of the particle sorting apparatus of this embodiment other than the above are the same as those of the above-described first embodiment.

<5. Third Embodiment>
Next, a particle sorting apparatus according to the third embodiment of the present disclosure will be described. In a particle sorter such as a cell sorter, when sorting particles such as cells, plate sorting using a substrate having a plurality of reaction sites (wells) (hereinafter referred to as a well plate) is sometimes performed. is there. There are various types of well plates used for this plate sorting, such as 6, 12, 24, 48, 96 and 384 wells, and the larger the number of wells, the better. The diameter of the well opening is reduced.

Therefore, when a plate having a large number of wells is used in the conventional particle sorting apparatus, it is difficult to accurately distribute target particles in the wells. Further, in the conventional particle sorting apparatus, when the diameter of the well becomes smaller, the droplets are more likely to hit the wall surface. Therefore, when the particles to be sorted are cells, the sorted cells are damaged and the survival of the cells. There is also the problem that the risk of the rate decreasing increases.

[Overall configuration of device]
FIG. 13 is a diagram schematically showing a configuration example of the particle fractionation device of the present embodiment, FIG. 14A is a diagram schematically showing the relationship between the side stream and the well plate, and FIG. 14B is a diagram showing conventional particles. It is a figure which shows typically the relationship between a side stream and a well plate in a fractionation apparatus. In FIG. 13, the same components as those of the particle sorting apparatus shown in FIG. 1 are designated by the same reference numerals, and detailed description thereof will be omitted.

As shown in FIG. 13, the particle sorting apparatus 31 of this embodiment includes a microchip 2, a vibrating element 3, a charging unit 4, deflection plates 5a and 5b, a waste liquid collection container 35, a well plate 36, and the like. Then, in the particle sorting apparatus 31 of the present embodiment, the well plate 36 is arranged so as to be inclined in a direction in which the incident angle θ of the fluid stream S with respect to the opening surface of the well 36a approaches 90°.

[Waste liquid collection container 35]
The waste liquid collecting container 35 collects liquid droplets containing particles or liquid droplets not containing particles that are not the target of sorting, and a general-purpose plastic tube, glass tube, or the like can be used for experiments. A drainage path for the collected droplets may be connected to the waste liquid collection container 35. In addition, it is preferable that the waste liquid collection container 35 is replaceably arranged in the apparatus at a position that does not hinder the collection of droplets by the well plate 36, particularly the movement of the well plate 36.

[Well plate 36]
The well plate 36 is used for a PCR method or the like, and a plurality of wells (reaction sites) 36a are formed on a substrate, and each well 36a contains one or more liquids containing specific particles. Drops are collected. Further, in the particle sorting apparatus 31 of the present embodiment, the well plate 36 is arranged so as to be inclined toward the fluid stream S. When the well plate 36 is arranged horizontally as in the conventional particle sorting apparatus shown in FIG. 14B, the droplet (fluid stream S) is incident from an oblique direction, so that the droplet may come off the opening of the well 36a (hit rate). Drop) or hit the side wall of the well 36a.

On the other hand, in the particle sorting apparatus 31 of the present embodiment shown in FIG. 14A, since the well plate 36 is tilted toward the side stream S, droplets easily enter the well 36a, and the well 36a is not easily removed. Hard to hit the side wall. As a result, the particle sorting apparatus 31 of the present embodiment can accurately sort the particles without causing damage, and particularly when the particles to be sorted are cells, the survival rate after sorting. Can be increased.

Here, the inclination angle of the well plate 36 is not particularly limited, but the well plate 36 is set so that the incident angle θ of the fluid stream S is approximately 90° or in the vicinity thereof with respect to the opening surface of the well 36a. Is preferably arranged in a tilted manner. Further, the number of wells 36a of the well plate 36 used in the particle sorting apparatus 31 of the present embodiment is not particularly limited, but the larger the number of wells 36a, the more remarkable the effect described above. Further, the shape of the well 36a is not limited, and various shapes such as a bottom surface having a flat surface or a curved surface can be used.

The method of tilting the well plate 36 is not particularly limited, but for example, there is a method of tilting the plate mounting portion of the plate holder holding the well plate 36 by a predetermined angle. FIG. 15 is a side view schematically showing a state in which a well plate is placed on a plate holder whose plate placing portion is inclined. As shown in FIG. 15, the plate mounting portion 37 a of the plate holder 37 is provided with an inclination of an angle α according to the angle of the fluid stream S, so that the well plate 36 mounted on the plate mounting portion 37 a becomes a fluid stream. It is possible to tilt toward.

Alternatively, the well plate 36 or the plate holder on which the well plate 36 is placed is placed on a stage inclined at an arbitrary angle, and the well plate 36 is inclined toward the fluid stream S by inclining the stage. Good.

[motion]
Next, the operation of the particle sorting apparatus 31 of this embodiment will be described. The particle sorting apparatus according to the present embodiment sequentially moves the well plate 36 by a moving mechanism so that the positions of the fluid stream S and the wells 36a of the well plate 36 coincide with each other, thereby allowing specific particles to be supplied to each well 36a. Distribute one or the desired number.

At that time, when the well plate 36 is arranged in an inclined manner, the horizontal distance between the wells 36a changes. Therefore, when the well plate 36 is moved in the same manner as in the case where the well plate 36 is arranged horizontally, the positions of the fluid stream S and the well 36a are changed. Error occurs. Therefore, in the particle sorting apparatus 31 of the present embodiment, for example, a movement control unit that controls the moving mechanism is provided, and the movement amount of the well plate 36 at the time of sorting is adjusted according to the inclination angle of the well plate 36. Accordingly, even if the well plate 36 is arranged in an inclined manner, the positions of the fluid stream S, the fluid stream S, and the well 36a can be made to coincide with each other, so that the fluid can be accurately separated.

In addition, when the cells are sorted, a buffer (buffer solution) is stored in advance in the well 36a. However, in the case of the well plate 36 having a shallow well 36a, if the well plate 36 is arranged in an inclined manner, the buffer may leak. is there. If the bottom of the well 36a is flat (flat bottom), or if the well 36a stores a small amount of buffer, the well plate 36 is inclined and the portion covered with the buffer is reduced.

Therefore, in the particle sorting apparatus 31 of the present embodiment, the inclination angle of the well plate 36 can be automatically adjusted according to the type of the well plate 36 and/or the amount of buffer stored. FIG. 16 is a flowchart showing an operation example of the particle sorting device 31 of the present embodiment. Specifically, as shown in FIG. 16, the user inputs the type of well plate (the number and shape of wells), or the data stored in the barcode or tag attached to the product is displayed. Read and determine the type of well plate. Further, the amount of buffer stored in the well is input by the user or automatically determined.

Next, for example, in the tilt control section provided in the apparatus, the tilt angle of the well plate is determined according to the type of well plate and/or the amount of buffer stored. Then, the well plate is tilted by the tilt angle adjusting mechanism so that the tilt angle is determined. After that, for example, the plate movement control unit provided in the apparatus determines the amount of movement of the well plate when the plate is tilted by a predetermined angle, and based on the result, the particles are fractionated while controlling the movement of the well plate.

In this way, by adjusting the inclination angle of the well plate according to the amount of buffer in which the type of well plate is stored, highly accurate sorting can be realized. When a well plate having a small number of wells is used, the diameter of the well is sufficiently large with respect to the positional accuracy of the fluid stream S, and therefore the merit of tilting the well plate is reduced. That is, the configuration of the present embodiment is particularly effective when using a well plate having a large number of wells and a small well diameter.

In the particle sorting apparatus of the present embodiment, since the well plate is arranged so as to be inclined toward the side stream, it is possible to accurately sort the particles to be sorted into a predetermined well without damaging them. ..

The particle sorting apparatus of the present embodiment can combine the above-described configuration with the configuration of the first embodiment, its modification, or the configuration of the second embodiment. For example, by combining the configuration of adjusting the charge application end time in the charging section according to the size of the particles contained in the liquid droplets, stable operation can be achieved even for liquid droplets containing large particles. An electric charge can be given, and the sorting accuracy can be further improved. In addition, for example, the break-off point can be maintained with high accuracy by controlling the drive voltage of the vibration element based on the state of the fluid stream in the vibration control unit together with the adjustment of the charge application end time. In addition to the charging of the droplets, the droplet formation can be stabilized.

Further, the present disclosure may also have the following configurations.
(1)
A charging portion that imparts a charge to at least a portion of the droplets ejected from the orifice that generates the fluid stream;
A charge control unit that adjusts the charge application end time in the charging unit according to the size of particles contained in the droplets;
A particle sorting device having.
(2)
The said charge control part is a particle fractionation apparatus as described in (1) which changes the timing of charge provision according to the size of the particle contained in the said droplet.
(3)
The said charge control part is a particle fractionation apparatus as described in (1) which changes charge provision time according to the size of the particle contained in the said droplet.
(4)
Irradiating particles flowing through the flow path with light, and having a forward scattered light detection unit for detecting forward scattered light generated from the particles by the light irradiation,
The said charge control part is a particle fractionation apparatus in any one of (1)-(3) which adjusts a charge provision completion time based on the detection result of the said front scattered light detection part.
(5)
The charge control unit, when the intensity of the forward scattered light detected by the forward scattered light detection unit is equal to or more than a preset threshold, than when the intensity of the forward scattered light is less than the threshold, the timing of charge application is The particle sorting apparatus according to (4), wherein the charging unit is controlled to be slow.
(6)
A delay amount calculation unit that calculates a delay amount of the charge application timing based on the intensity of the forward scattered light detected by the forward scattered light detection unit,
The particle sorting apparatus according to (4) or (5), wherein the charge control unit controls the charging unit so that the charge application timing is delayed by the delay amount calculated by the delay amount calculation unit.
(7)
The charge control unit, when the intensity of the forward scattered light detected by the forward scattered light detection unit is equal to or greater than a preset threshold value, the charge application time is longer than when the intensity of the forward scattered light is less than the threshold value. (4) The particle sorting apparatus according to (4), wherein the charging unit is controlled so that
(8)
It has an application time calculation unit for calculating the charge application time based on the intensity of the forward scattered light detected by the forward scattered light detection unit,
The particle control apparatus according to (4) or (7), wherein the charge control unit controls the charging unit so that the charge is applied for the time period calculated by the application time calculation unit.
(9)
The orifice is formed in a replaceable microchip,
The particle according to any one of (1) to (8), wherein the charging unit includes a charging electrode arranged in contact with a sheath liquid and/or a sample liquid flowing in a channel provided in the microchip. Preparative device.
(10)
The particle sorting apparatus according to any one of (1) to (8), wherein the orifice is formed in a flow cell.
(11)
Applying a charge to at least a portion of the droplets ejected from the orifice that produces the fluid stream,
A particle sorting method for adjusting a charge application end time according to the size of particles contained in the droplet.
(12)
A program that causes the charge control unit of the particle sorting apparatus to perform the function of adjusting the charge application end time according to the size of the particles contained in the droplets discharged from the orifice that generates the fluid stream.

Furthermore, the present disclosure may also be configured as below.
(1)
A charging portion that imparts a charge to at least a portion of the droplets ejected from the orifice that generates the fluid stream;
A deflection plate for changing the traveling direction of the charged droplets,
A base material having a plurality of recesses, in which the droplets containing specific particles are collected,
Have
A particle sorter in which the substrate is tilted towards a fluid stream formed by droplets containing the particular particles.
(2)
A base material holder for holding the base material,
The particle sorting device according to (1), wherein the base material mounting portion of the base material holder is inclined so that the base material is inclined.
(3)
The particle sorting apparatus according to (1) or (2), wherein the base material is inclined and arranged so that an incident angle of the fluid stream is approximately 90° with respect to an opening surface of the recess.
(4)
Having a tilt angle adjusting portion of the base material,
The said particle|grain sorting apparatus in any one of (1)-(3) which adjusts the inclination|tilt angle of the said base material based on the information of at least one of the kind and state of the said base material.
(5)
A base material moving mechanism for changing the position of the base material such that specific particles are distributed to each concave portion one by one or by a desired number;
A base material movement control unit that controls the movement of the base material by the base material moving mechanism,
The said base material movement control part is a particle fractionation apparatus in any one of (1)-(4) which adjusts the movement amount of the said base material according to the inclination angle of the said base material.
(6)
The particle sorting apparatus according to any one of (1) to (5), further including a charge control unit that adjusts a charge application end time in the charging unit according to a size of particles included in the droplet.
(7)
An imaging device for acquiring an image of the fluid and droplets at a position where the fluid discharged from an orifice generating a fluid stream is dropletized;
Based on the state of the fluid in the image and/or the state of satellite droplets present between the location where the fluid is dropletized and the droplet closest to the location where the fluid is dropletized A vibration control unit that controls the drive voltage of the vibration element that gives vibration,
The particle fractionation device according to any one of (1) to (6), which further comprises:

It should be noted that the effects described in the present specification are merely examples and are not limited, and may have other effects.

1, 11, 31 Particle Sorting Device 2 Microchip 3 Vibration Element 4 Charging Section 5a, 5b Deflection Plates 6a to 6c Recovery Container 7 Charging Control Section 8 Light Detection Section 9 Delay Amount Calculation Section 10 Application Time Calculation Section 12 Imaging Element ( camera)
13, 42 Voltage Supply Section 14 Vibration Control Section 15 Position Adjustment Mechanism 21 Orifice 22 Sample Inlet 23 Sheath Inlet 24 Suction Outlet 35 Waste Liquid Collection Container 36 Well Plate 36a Well 37 Plate Holder 41 Electrode 71 Delay Amount Calculation Section 72 Application Time Calculation Section S fluid stream

Claims (11)

A charging portion that imparts a charge to at least a portion of the droplets ejected from the orifice that generates the fluid stream;
A normal mode in which the charging unit is controlled at least at the first charge application timing or the first charge application time, and the second charge application timing delayed from the first charge application timing or the first charge application time. A charge control unit that adjusts the charge application end time in the charging unit based on a large particle mode that controls the charging unit with a second charge application time that is increased from the charge application time,
A particle sorting device having a. The particle sorting apparatus according to claim 1, wherein the charge control unit implements the normal mode or the large-diameter particle mode according to a user's selection. Irradiating particles flowing through the flow path with light, and having a forward scattered light detection unit for detecting forward scattered light generated from the particles by the light irradiation,
When the large particle mode is selected , the charge control unit adjusts the second charge application timing or the second charge application time based on the detection result of the forward scattered light detection unit. Item 2. The particle sorting apparatus according to item 2 . The charge control unit, when the intensity of the forward scattered light detected by the forward scattered light detection unit is equal to or more than a preset threshold, than when the intensity of the forward scattered light is less than the threshold, the timing of charge application is The particle sorting apparatus according to claim 3 , wherein the charging unit is controlled to be slow. A delay amount calculation unit that calculates a delay amount at the timing of charge application based on the intensity of the forward scattered light detected by the forward scattered light detection unit,
The particle sorting apparatus according to claim 3 , wherein the charge control unit controls the charging unit so that the timing of charge application is delayed by the delay amount calculated by the delay amount calculation unit. The charge control unit, when the intensity of the forward scattered light detected by the forward scattered light detector is equal to or greater than a preset threshold value, the charge application time is longer than when the intensity of the forward scattered light is less than the threshold value. The particle sorting apparatus according to claim 3 , wherein the charging unit is controlled so that It has an application time calculation unit for calculating the charge application time based on the intensity of the forward scattered light detected by the forward scattered light detection unit,
The particle sorting apparatus according to claim 3 , wherein the charging control unit controls the charging unit so that the charges are charged for the time period calculated by the charging time period calculation unit. The orifice is formed in a replaceable microchip,
The particle sorting apparatus according to claim 1, wherein the charging unit includes a charging electrode arranged in contact with a sheath liquid and/or a sample liquid flowing in a flow path provided in the microchip. The particle sorting apparatus according to claim 1, wherein the orifice is formed in a flow cell. Applying a charge to at least a portion of the droplets ejected from the orifice that produces the fluid stream,
A normal mode in which at least the first charge application timing or the first charge application time controls the charging unit, and a second charge application timing delayed from the first charge application timing or the first charge A particle sorting method for adjusting an end time of charge application based on a large-diameter particle mode in which a charging part is controlled by a second charge application time increased from the application time. A normal mode in which an electric charge is applied to at least a part of a droplet discharged from an orifice that generates a fluid stream, and the charging unit is controlled at least at the timing of the first charge application or the first charge application time; Based on the second charge application timing delayed from the one charge application timing or the large particle mode for controlling the charging part at the second charge application time increased from the first charge application time , and A program that causes the charge control unit of the particle sorting apparatus to execute the function of adjusting the charge application end time.