JP6508265B2 - PARTICLE SEPARATING DEVICE AND PARTICLE SEPARATING METHOD - Google Patents

Description

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

  Heretofore, an optical measurement method using flow cytometry (flow cytometer) has been used for analysis of biologically relevant particles such as cells, microorganisms and liposomes. A flow cytometer is an apparatus that irradiates light to particles flowing in a flow channel formed in a flow cell, a microchip, or the like, and detects and analyzes fluorescence or scattered light emitted from each particle.

  Some flow cytometers have a function to separate and collect only particles having specific characteristics based on the analysis results, and in particular, an apparatus for separating cells is called "cell sorter". . As a sorting method of a cell sorter, a droplet charging method in which droplets containing particles are charged and separated is mainly adopted (for example, see Patent Document 1). In a droplet charging type device, fluid discharged from a flow cell or microchip is formed into droplets, positive (+) or negative (-) charges are applied to the droplets, and the traveling direction is determined by a deflection plate or the like. Collect in a specified container by changing.

  However, the separation method such as the droplet charging method, which forms droplets, has a problem that it is susceptible to changes in the measurement environment and fluid pressure fluctuations. Therefore, conventionally, a particle sorting apparatus for sorting in a microchip has also been proposed (see Patent Document 2). Since the particle sorting apparatus described in Patent Document 2 sucks the particles to be recovered to the negative pressure suction portion in the microchip and separates the particles, there is no need for dropletization or charging, and the particles are damaged. It is possible to carry out the separation at high speed and stably.

JP, 2009-145213, A JP, 2012-127922, A

  In the conventional particle sorting apparatus, generally, it is controlled to perform an operation of acquiring particles to be collected after a predetermined time from the detection time in the light detection unit. The time from detection to acquisition is preset based on the fluid pressure, the distance from the detection position to the dispensing position, and the like. However, in the control method in which the arrival time is fixed in this way, there is a problem that when the flow rate of particles fluctuates, the purity and the acquisition rate of the recovered material decrease.

  On the other hand, in the device described in Patent Document 1, in order to prevent a decrease in purity due to fluctuations in particle flow velocity, the movement velocity is detected for each particle, and the timing for applying charge to each particle is determined based on the movement velocity. I have control. However, in the case of the charged droplet method, it is only necessary to judge to which droplet each particle belongs, but in the case of an apparatus which performs sorting in a microchip, in addition to the attributes of the adjacent particles, fluid mechanical characteristics Need to be considered. Here, the “attribute of particle” is, for example, whether or not the particle is a particle to be separated, and the “fluid mechanical property” is a reverse flow or the like generated at the rise of the pulse signal of the acquisition operation.

  Moreover, although control is performed on droplets in the charged droplet method, in an apparatus for performing separation in a microchip as described in Patent Document 2, it is necessary to control individual particles. . Furthermore, the charged droplet method and the method for sorting within the microchip differ in the route to the acquisition position and the factors that affect the arrival of particles. For the above reasons, the technique described in Patent Document 1 can not simply be applied to the apparatus described in Patent Document 2 which performs sorting in a microchip.

  Therefore, the present disclosure is mainly intended to provide a particle sorting apparatus and a particle sorting method capable of efficiently sorting particles in a microchip.

In the particle sorting device according to the present disclosure, a first light irradiation unit that irradiates the first irradiation light to the particles flowing through the flow path, and the second irradiation light at a position different from the first irradiation light to the particles From the detection time difference between the light originating from the first irradiation light and the light originating from the second irradiation light, the second light irradiation part for irradiation, the light detection part for detecting the light emitted from the particles, and the front and back A separation control unit that calculates the time difference between the particles reaching the separation unit communicating with the flow path, and controls the separation of the particles based on the difference in arrival time of the particles before and after the separation mode and the calculated mode. And the flow path and the sorting unit are provided in the microchip.
The second irradiation light may be different in wavelength from the first irradiation light.
The particle sorting device according to the present disclosure individually calculates the time for each particle to reach the sorting portion from the detection time difference between the light originating from the first irradiation light and the light originating from the second irradiation light. A calculation unit may be further included. In that case, the calculation unit may calculate, from the time, the time difference between the front and back particles reaching the sorting unit.
The particles may be biorelevant particles.
The particle sorting device according to the present disclosure may further include a sample liquid introduction channel into which the sample liquid containing the particles is introduced, and a pair of sheath liquid introduction channels into which the sheath liquid is introduced. Good.
The first light irradiation unit may include two or more light sources. In that case, the two or more light sources may emit light of different wavelengths.
The light detection unit may be configured of any one or more area imaging elements selected from the group consisting of photomultiplier tubes (PMTs), CCDs, and CMOS elements.
The light detection unit may detect any one or more lights selected from the group consisting of forward scattered light, side scattered light, Rayleigh scattered light, and Mie scattered light.
The sorting control unit may determine that the particles are obtained or not obtained according to the sorting mode selected by the user. In that case, the separation control unit may determine that the particles whose arrival time difference is less than or equal to the threshold value are not collected.
The fractionating unit may have a negative pressure suction unit in communication with the flow path. In that case, the separation control unit may control the operation of the negative pressure suction unit based on the data of each particle detected by the light detection unit and the time calculated by the calculation unit. .
The separation control unit controls the timing of collecting the particles in the separation unit based on the data of each particle detected by the light detection unit and the time calculated by the calculation unit. Good.

Particle analysis system according to the present disclosure includes a first light irradiation part for irradiating the first irradiation light to particles passing through the flow channel, the second irradiation light at a position different from the first irradiation light to the particles A microparticle measurement apparatus comprising at least a second light irradiation unit to be irradiated and a light detection unit to detect light emitted from the particles, light derived from the first irradiation light and the second irradiation light From the detection time difference of the derived light, calculate the time difference for the front and back particles to reach the separation part communicating with the flow path, and based on the separation time of the separation mode and the calculated before and after particles, At least a control unit having at least a sorting control unit for controlling the sorting, wherein the flow path and the sorting unit are provided in the microchip.
In the particle sorting method according to the present disclosure, a first light irradiation step of irradiating the particles flowing through the flow channel with the first irradiation light, and the second irradiation at the position different from the first irradiation light to the particles From the second light irradiation process of irradiating light, the light detection process of detecting light emitted from the particles, and the detection time difference between the light derived from the first irradiation light and the light derived from the second irradiation light, Separation control which calculates the time difference between the front and back particles reaching the separation part communicating with the flow path, and controls the separation of the particles based on the difference in arrival time between the separation mode and the calculated before and after particles. And the flow path and the sorting unit are provided in the microchip.

  According to the present disclosure, the sorting control unit determines whether to collect the particles based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit. Therefore, the particle acquisition performance can be improved.

It is a figure showing typically the composition of the particle sorting device concerning a 1st embodiment of this indication. It is a figure which shows the detection data in the light detection part 7 shown in FIG. It is a block diagram which shows the circuit structure of the arrival time calculation part 8 of the particle | grain separation apparatus which concerns on the modification of 1st Embodiment of this indication, and the distribution control part 9. As shown in FIG. A and B are figures which show the process in the event detection circuit shown in FIG. A and B are figures which show the distance in the gating circuit shown in FIG. It is a figure which shows operation | movement of acquisition priority mode. A and B are figures which show the detection data in case particle | grains adjoin. It is a figure which shows operation | movement of purity priority mode.

Hereinafter, an embodiment for carrying out the present disclosure will be described in detail with reference to the attached drawings.
In addition, this indication is not limited to each embodiment shown below. The description will be made in the following order.

1. First Embodiment (Example of Particle Sorting Device Having Sorting Control Unit)
2. Modification of First Embodiment (Example of Particle Sorting Device Having Mode Switching Function)

<1. First embodiment>
First, a particle sorting device according to a first embodiment of the present disclosure will be described. FIG. 1 is a view showing a schematic configuration of a particle sorting device according to a first embodiment of the present disclosure. FIG. 2 is a view showing detection data in the light detection unit 7.

[Overall configuration of device]
As shown in FIG. 1, the particle sorting apparatus 1 of the present embodiment separates and recovers the particles 10 based on the result of analysis by an optical method or the like. The particle sorting apparatus 1 includes, for example, a flow path 1, a sorting unit 2, an excitation light irradiating unit 3, a speed detecting light irradiating unit 4, a light detecting unit 7, an arrival time calculating unit 8, a sorting control unit 9, and the like. Is equipped.

[About particle 10]
The particles 10 to be analyzed and separated by the particle sorting apparatus 1 of the present embodiment include cells, bio-related particles such as microorganisms and ribosomes, or synthetic particles such as latex particles, gel particles and particles for industrial use, etc. included.

  Biologically relevant particles include chromosomes constituting various cells, ribosomes, mitochondria, organelles (cellular organelles) and the like. The cells also include plant cells, animal cells and blood cells. Furthermore, microorganisms include bacteria such as E. coli, viruses such as tobacco mosaic virus, and fungi such as yeast. The biorelevant particle may include biorelevant polymers such as nucleic acids, proteins, and complexes thereof.

  On the other hand, as particles for industrial use, those formed of, for example, an organic polymer material, an inorganic material, or a metal material can be mentioned. As the organic polymer material, polystyrene, styrene / divinylbenzene, polymethyl methacrylate and the like can be used. Further, as the inorganic material, glass, silica, magnetic material and 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, mass, etc. are not particularly limited.

[Flow path 1]
The flow path 1 is formed in the microchip, and a liquid (sample liquid) containing particles 10 to be separated and to be separated is introduced. Here, the microchip provided with the flow path 1 can be formed of glass or various plastics (PP, PC, COP, PDMS, etc.). Further, the material of the microchip is transparent to the light irradiated from the excitation light irradiation unit 3 and the speed detection light irradiation unit 4 and has less autofluorescence and less optical error due to small wavelength dispersion. It is desirable to use a material.

  On the other hand, the channel 1 can be formed by wet etching or dry etching of a glass substrate, or by nanoimprinting, injection molding, or machining of a plastic substrate. The microchip can be formed, for example, by sealing a substrate obtained by molding the channel 1 or the like with a substrate of the same material or a different material.

  Although FIG. 1 shows only a portion of the flow path 1 to which excitation light and speed detection light are irradiated, the sample liquid introduction flow into which the sample liquid containing the particle 10 is introduced upstream of this A channel and a pair of sheath fluid introduction channels into which the sheath fluid is introduced may be provided. In this case, the sheath fluid introduction channel joins the sample fluid introduction channel from both sides, and the channel 1 is provided downstream of the junction point. Then, in the flow channel 1, the sheath flow is surrounded by the sheath flow to form a laminar flow, and the liquid flows therethrough, and the particles 10 in the sample liquid are arranged in approximately one row with respect to the flow direction. It flows side by side.

[Distribution Division 2]
The sorting unit 2 is for sorting the particles 10 to be collected, and is formed in the microchip. The fractionating unit 2 communicates with the downstream end of the flow passage 13 and includes a suction flow passage 21 and a negative pressure suction unit 22. The configuration of the negative pressure suction unit 22 is not particularly limited as long as it can suction the fine particles to be collected at a predetermined timing. For example, the negative pressure suction unit may be an actuator (not shown) or the like. The 22 volumes can be configured to be expandable at any timing.

[Excitation light irradiation unit 3]
The excitation light irradiator 3 is provided with a light source 31 for generating excitation light such as laser light, an optical system 32 for shaping a spot shape, a mirror 33 and the like. Then, for example, the particle 10 flowing in the flow path 1 formed in the microchip is irradiated with excitation light. In addition, although the case where one light source 31 is shown as an example in FIG. 1, this indication is not limited to this, Two or more light sources 31 may be provided, In that case, each Light of different wavelength may be emitted from the light source 31.

[Light irradiator 4 for speed detection]
The speed detection light irradiator 4 is provided with a light source 41 for generating the speed detection light, an optical system 42 for shaping the spot shape, a mirror 43 and the like. Then, for example, the particle 10 flowing in the flow path 1 formed in the microchip is irradiated with the velocity detection light at a position different from the excitation light described above. The speed detection light may be light of the same wavelength as the excitation light, but it is preferable to use light having a wavelength different from that of the excitation light from the viewpoint of simplification of the apparatus configuration.

[Light detection unit 7]
The light detection unit 7 detects light (scattered light, fluorescence, etc.) generated from the particles 10 flowing through the flow channel 1, and the zero-order light removing member 71, mirrors 72a to 72d, and light detectors 73a to 73 73d and so on. For the photodetectors 73a to 73d, for example, an area imaging device such as a photomultiplier tube (PMT) or a CCD or a CMOS device can be used.

  In the light detection unit 7, for example, forward scattered light derived from the excitation light by the light detector 73a, scattered light derived from the speed detection light by the light detector 73b, and fluorescence by the light detectors 73c and 73d, respectively. To detect. The light to be detected by the light detection unit 7 is not limited to these, and side scattered light, Rayleigh scattering, Mie scattering, or the like may be detected. Then, the light detected by the light detection unit 7 is converted into an electrical signal.

[Arrival time calculation unit 8]
From the detection time difference between the light derived from the excitation light and the light derived from the speed detection light, the time for each particle 10 to reach the separation unit 2 communicating with the flow path is individually calculated. Although the calculation method of arrival time is not particularly limited, for example, as shown in FIG. 2, forward scattered light (data of Ch1) derived from the excitation light detected by the light detection unit 7 and speed detection The arrival time of each particle 10 is calculated from the detection time difference of forward scattered light (data of Ch2) derived from the use light.

  Here, the arrival time to the fractionating unit 2 can be calculated, for example, by a simple linear approximation formula shown in the following Equation 1. Note that L1 in the following formula 1 is the distance between the excitation light irradiation position and the speed detection light irradiation position, and L2 is the distance from the speed detection light irradiation position to the suction flow channel 21 of the sorting unit 2 (see FIG. 1) ). Moreover, T1 in the following formula 1 is a detection time of light derived from excitation light, T2 is a detection time of light derived from speed detection light, and (T1-T2) is a difference between these detection times (see FIG. 2).

  Note that the method of calculating the arrival time to the sorting unit 2 is not limited to the linear calculation method shown in Equation 1 above, and another calculation method such as polynomial approximation or a look-up table may be used.

[Distribution control unit 9]
The separation control unit 9 controls the separation of the particles 10, and based on the data of each particle 10 detected by the light detection unit 7 and the arrival time calculated by the arrival time calculation unit 8, It is determined whether to collect 10 or not. The fractionating control unit 9 calculates, for example, the arrival time difference between the front and back particles 10, and determines that the calculated arrival time difference is equal to or less than a preset threshold value as "non-collection". This makes it possible to prevent the particles before and after the particles to be collected from being caught and acquired when the particles 10 flow close to each other.

  Further, the separation control unit 9 controls the timing of collecting the particles 10 in the separation unit 2 by, for example, controlling the operation of the negative pressure suction unit 22 based on the determination result described above. As a result, it is possible to improve the acquisition accuracy of the target particles and to perform the separation with high purity and acquisition rate.

[Operation]
Next, the operation of the particle sorting device of the present embodiment will be described. When the particles are separated by the particle separation device of the present embodiment, the sample liquid containing particles to be separated is introduced into the sheath inlet, respectively, to the sample inlet provided in the microchip. . Then, the particle 10 flowing through the flow channel 1 is irradiated with excitation light, and the particle 10 is irradiated with velocity detection light at a position different from the excitation light. At this time, as shown in FIG. 1, the excitation light and the speed detection light may be condensed by one condensing lens 5 and may be irradiated to the particle 10, but it is condensed by different condensing lenses. It is also good.

  Next, the detection unit 7 detects the light emitted from each particle 10, and the arrival time calculation unit 8 separates the separation unit from the detection time difference between the light derived from the excitation light and the light derived from the speed detection light. The time for each particle 10 to reach 2 is calculated individually. At this time, as shown in FIG. 1, the light derived from the excitation light and the light derived from the speed detection light are condensed by one condensing lens 6 and collected in the zero-order light removing member 71 of the detection unit 7. Although it may be lighted, it may be collected by separate condensing lenses.

  Thereafter, in the separation control unit 9, whether the particles 10 are recovered from the optical characteristic data of each particle 10 detected by the detection unit 7 and the arrival time to the separation unit 2 calculated by the arrival time calculation unit 8 Decide whether or not. Then, based on the determination result, the sorting control unit 9 controls the timing of collecting the particles 10 in the sorting unit 2. For example, when the fractionating unit 2 includes the negative pressure suction unit 22 communicating with the flow path 1, the fractionating control unit 9 controls the operation of an actuator or the like provided in the negative pressure suction unit 22.

  As described above in detail, in the particle sorting apparatus according to the present embodiment, the arrival time to the sorting unit is calculated for each particle, and the arrival time to the sorting unit as well as the optical characteristic data of each particle is calculated. In addition, it is determined whether or not to collect particles. This makes it possible to separate particles with high purity or a high acquisition rate regardless of the flowing position or flowing state of particles. As a result, the acquisition performance can be improved as compared to the conventional particle sorting apparatus.

  Further, the particle sorting apparatus of the present embodiment calculates the arrival time for each particle, and therefore, is not easily affected by the flow rate change due to the environmental temperature change or the remaining amount of the supply tank. As a result, since it is not necessary to perform flow control with high accuracy, a low-cost pressure control device can be adopted, and it becomes possible to simplify flow path component management and assembly accuracy management, and reduce manufacturing costs. be able to.

<2. Modification of First Embodiment>
Next, a particle sorting device according to a modification of the first embodiment of the present disclosure will be described. In the particle sorting device of the present modification, the user can select whether to prioritize "purity" or "acquisition ratio" when determining whether or not to recover.

  FIG. 3 is a block diagram showing a circuit configuration of the arrival time calculation unit 8 and the separation control unit 9 of the particle sorting device of the present modification. 4A and 4B are diagrams showing processing in the event detection circuit, and FIGS. 5A and 5B are diagrams showing distance in the gating circuit. The separation in the “purity priority mode” or the “acquisition ratio priority mode” can be realized, for example, by the circuit having the configuration shown in FIG.

[Event detection circuit]
The event detection circuit is triggered by the detection signals of Ch1 and Ch2, reads the waveform of each Ch, and calculates the width, height, and area shown in FIG. 4A. Then, for the detection data of Ch1 regarding the forward scattered light derived from the excitation light and the detection data of Ch2 regarding the forward scattered light derived from the light for speed detection, the time of the waveform center is calculated and used as the detection time.

  Then, as shown in FIG. 4B, in the event detection circuit, Ch1 (forward scattered light derived from excitation light) and Ch2 (forward scattered light derived from speed detection light) acquired in time series for each particle 10 The detection signal of each particle is associated, and the detection data (event) of each particle is packetized. The packet includes an item updated as the subsequent processing proceeds, and Flag is basically 1/0, corresponding to acquisition / non-acquisition determined by each logic. The detection time can also use the trigger time of Ch1 and Ch2.

Arrival time calculation circuit
The arrival time calculation circuit uses the detection times (T1, T2) of Ch1 and Ch2 to calculate the arrival time from the above equation 1 or the like, and uses it as the "Sorting Time" of the event packet.

[Gating circuit]
The gating circuit determines “acquisition / non-acquisition” of the particle 10 based on a preset threshold value, and sets “Gate Flag” of the event packet. For example, before starting the gating acquisition operation, the histogram chart shown in FIG. 5A or the 2D chart shown in FIG. Particle groups are specified by, for example, geometric shapes.

  The parameter (threshold value) for determining “acquisition / non-acquisition” may be any of the width, height, and area of the detection data acquired in each Ch, or may be a combination of these.

[Output queue circuit]
The output queue circuit rearranges detection data (events) of each particle 10 in the order of arrival at the separation unit based on the arrival time of the separation unit (“Sorting Time”). Thereafter, in accordance with the sorting mode selected by the user, such as "purity priority" or "acquisition ratio priority", "acquisition / non-acquisition" determination is performed. Then, based on the result, "Sort Flag" is set.

  When the particles 10 flow in close proximity, it is possible that the particles 10 before and after may also be involved in one acquisition operation, and the plurality of particles 10 may be collected in the separation unit 2. And, in the “purity priority mode” and the “acquisition rate priority mode”, the determination method of “acquisition / non-acquisition” when the particles 10 are close to each other is different. FIG. 6 is a diagram showing the operation in the acquisition priority mode. 7A and 7B are diagrams showing detection data in the case where particles are close to each other. Furthermore, FIG. 8 is a diagram showing the operation of the purity priority mode.

  The “acquisition priority mode” is a mode in which the number of acquired particles is increased even if the purity of the captured particles decreases, and as shown in FIG. Collect the particles. On the other hand, the "purity priority mode" is a mode to increase the purity of capture particles, and when the acquisition particles and non-acquisition particles come close, they are dared to be captured to prevent them from being captured together. Is judged as "not acquired".

  In particular, in the case of the "purity priority mode", as shown in FIG. 8, when the detection data (event) of the particle 10 detected later is close to the previous particle 10, "acquisition / non-generation of the previous event" is performed. "Acquisition" also needs to be judged again. Here, ΔT1 shown in FIG. 7A is a set value, which is a time for which the next particle is involved (T1 = Tn + ΔT1). Further, ΔT2 is also a set value, which is a time for which the immediately preceding particle is involved (T2 = Tn + ΔT2).

[Output timing generation circuit]
The output timing generation circuit reads the time (Sorting time) of the event acquired first in the output queue, compares it with the Clock Counter value, and generates an output timing signal at that time.

[Output signal generation circuit]
The output signal generation circuit detects an output timing signal, and outputs a waveform signal that controls the actuation device of the separation unit 2.

  In the particle sorting apparatus of this modification, the user can select whether to prioritize "purity" or "acquisition ratio" when determining whether or not to collect the particles, so that the purpose is It will be possible to carry out the sorting according to the The remaining configuration and effects of the present modification are similar to those of the aforementioned first embodiment.

Further, the present disclosure can also be configured as follows.
(1)
An excitation light irradiation unit that irradiates excitation light to particles flowing through the flow path;
A speed detection light irradiator for irradiating the particle with speed detection light at a position different from the excitation light;
A light detection unit that detects light emitted from the particles;
An arrival time calculation unit that individually calculates the time for each particle to reach the separation unit communicating with the flow path from the detection time difference between the light derived from the excitation light and the light derived from the speed detection light;
A separation control unit that controls the separation of the particles;
The flow path and the separation unit are provided in a microchip,
The separation control unit determines whether to collect the particles based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit. apparatus.
(2)
The particle sorting device according to (2), wherein the fractionating control unit calculates an arrival time difference between front and back particles, and determines that particles having the arrival time difference less than or equal to a threshold are not collected.
(3)
The particle sorting apparatus according to (1) or (2), wherein the light for velocity detection has a wavelength different from that of the excitation light.
(4)
The particle sorting apparatus according to (3), wherein the arrival time calculation unit calculates the arrival time of each particle from a detection time difference between scattered light derived from the excitation light and scattered light derived from the speed detection light.
(5)
The particle separation apparatus according to any one of (1) to (4), wherein the excitation light irradiation unit includes two or more light sources that emit light of different wavelengths.
(6)
The particle sorting apparatus according to any one of (1) to (5), wherein the sorting unit has a negative pressure suction unit in communication with the flow path.
(7)
The separation control unit controls the operation of the negative pressure suction unit based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit (6). Particle separation device as described.
(8)
The separation control unit controls timing of collecting the particles in the separation unit based on data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit. The particle | grain separation apparatus in any one of (1)-(7).
(9)
An excitation light irradiation step of irradiating excitation light to particles flowing through a flow path provided in the microchip;
A speed detection light irradiation step of irradiating the particles with speed detection light at a position different from the excitation light;
A light detection step of detecting light emitted from the particles;
From the difference in detection time between the light derived from the excitation light and the light derived from the speed detection light, the time for each particle to reach the separation section provided in the microchip and communicated with the flow path is individually determined. Attainment time calculation process to calculate
A separation control step of determining whether to collect the particles based on data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step;
Method of particle separation having.
(10)
The method for separating particles according to (9), wherein the separation control step calculates an arrival time difference between front and back particles, and determines that the arrival time difference is less than or equal to a threshold value as non-recovery.
(11)
The particle sorting method according to (9) or (10), wherein light having a wavelength different from that of the excitation light is used as the speed detection light.
(12)
The arrival time calculation step calculates the arrival time of each particle from the detection time difference between the scattered light derived from the excitation light and the scattered light derived from the speed detection light (11).
(13)
The said excitation light irradiation process radiate | emits the light of a respectively different wavelength from 2 or more light sources, The particle | grain separation method in any one of (9)-(12).
(14)
The sorting unit has a negative pressure suction unit in communication with the flow path,
The fractionating control step controls the operation of the negative pressure suction unit based on the data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step (9) to (13) The particle sorting method according to any one of the above.
(15)
The separation control step controls the timing of collecting the particles in the separation part based on the data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step (9 The particle | grain separation method in any one of-).

DESCRIPTION OF SYMBOLS 1 flow path 2 separation part 3 excitation light irradiation part 4 light irradiation part 5 for speed detection 5 6 objective lens 7 light detection part 8 arrival time calculation part 9 separation control part 10 particle 21 suction flow path 22 negative pressure suction part 31 , 41 light source 32, 42 optical system 33, 43, 72a to 72d mirror 71 0th order light removing member 73a to 73d photodetector

Claims (16)

A first light irradiation unit that irradiates the first irradiation light to particles flowing through the flow path;
A second light irradiator for irradiating the particles with a second irradiation light at a position different from the first irradiation light;
A light detection unit that detects light emitted from the particles;
From the detection time difference between the light derived from the first irradiation light and the light derived from the second irradiation light, the time difference for the particles before and after to reach the separation part communicating with the flow path is calculated, and the separation mode and A separation control unit that controls the separation of the particles based on the calculated arrival time difference between before and after the particles;
Have at least
The flow path and the separation unit are provided in a microchip ,
The particle sorting apparatus , wherein the sorting unit has a negative pressure suction unit in communication with the flow path .   The particle sorting device according to claim 1, wherein the second irradiation light is different in wavelength from the first irradiation light. A calculation unit that individually calculates the time for each particle to reach the separation unit from the detection time difference between the light derived from the first irradiation light and the light derived from the second irradiation light,
The particle sorting apparatus according to claim 1, further comprising   The particle sorting device according to claim 3, wherein the calculation unit calculates a time difference in which particles before and after reach the sorting unit from the time.   The particle sorting device according to any one of claims 1 to 4, wherein the particles are biorelevant particles. A sample liquid introduction channel into which a sample liquid containing the particles is introduced;
A pair of sheath fluid introduction channels into which the sheath fluid is introduced;
The particle sorting device according to any one of claims 1 to 5, further comprising   The particle sorting apparatus according to any one of claims 1 to 6, wherein the first light irradiation unit includes two or more light sources.   The particle sorting device according to claim 7, wherein the two or more light sources emit light of different wavelengths.   The light detection unit according to any one of claims 1 to 8, wherein the light detection unit includes one or more area imaging devices selected from the group consisting of a photo multiplier tube (PMT), a CCD, and a CMOS device. Particle separation device.   10. The light detection unit according to any one of claims 1 to 9, wherein any one or more lights selected from the group consisting of forward scattered light, side scattered light, Rayleigh scattered light, and Mie scattered light are detected. Particle separation device as described.   The particle sorting device according to any one of claims 1 to 10, wherein the sorting control unit determines that the particles are obtained or not obtained according to a sorting mode selected by a user.   The particle sorting device according to claim 11, wherein the sorting control unit determines that particles having the arrival time difference equal to or less than a threshold are not collected. The distribution control unit according to claim 3 or 4 , wherein the operation of the negative pressure suction unit is controlled based on data of each particle detected by the light detection unit and the time calculated by the calculation unit. Particle separation device as described.   The separation control unit controls timing of collecting the particles in the separation unit based on data of each particle detected by the light detection unit and the time calculated by the calculation unit. The particle | grain separation apparatus of claim 3 or 4. A first light irradiator for irradiating the particles flowing through the channel with the first irradiation light, a second light irradiator for irradiating the particles at a position different from the first irradiation light, and the second light irradiator; A microparticle measurement device having at least a light detection unit that detects light emitted from particles;
From the detection time difference between the light derived from the first irradiation light and the light derived from the second irradiation light, the time difference for the particles before and after to reach the separation part communicating with the flow path is calculated, and the separation mode and A control device having at least a separation control unit for controlling the separation of the particles based on the calculated arrival time difference between the front and back particles;
Have at least
The flow path and the separation unit are provided in a microchip ,
The particle analysis system , wherein the sorting unit has a negative pressure suction unit in communication with the flow path . A first light irradiation step of irradiating the particles flowing through the flow path with the first irradiation light;
A second light irradiation step of irradiating the particles with a second irradiation light at a position different from the first irradiation light;
A light detection step of detecting light emitted from the particles;
From the detection time difference between the light derived from the first irradiation light and the light derived from the second irradiation light, the time difference for the particles before and after to reach the separation part communicating with the flow path is calculated, and the separation mode and A separation control step of controlling the separation of the particles based on the calculated arrival time difference between before and after the particles;
Have at least
The flow path and the separation unit are provided in a microchip ,
The particle sorting method , wherein the sorting portion has a negative pressure suction portion in communication with the flow path .

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