JP2016095221A - Particle analyzing device and particle analyzing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle analyzing device that reduces noise on a signal obtained on the basis of light emitted from particles that went through light irradiation.SOLUTION: A particle analyzing device includes: a first irradiation unit for irradiating first light to particles passing through a channel at a first position on the channel; a second irradiation unit for irradiating second light to the particles at a second position downstream of the first position; a light detection unit for obtaining an optical signal emitted by the particles and outputting a voltage signal; and a signal processing unit for determining an acquisition point on the basis of the time axis of a second voltage signal output by the light detection unit on the basis of the second light irradiation by the second irradiation unit on the basis of a first voltage signal output on the basis of the first light irradiation by the first irradiation unit and flow rate of the particles.SELECTED DRAWING: Figure 2

Description

  The present technology relates to a particle analysis apparatus and a particle analysis method. More specifically, the present invention relates to a particle analyzer and a particle analysis method for irradiating particles flowing through a flow path with light.

  In recent years, a device has been developed to measure the microparticles and analyze the measured microparticles in the process of passing biological microparticles such as cells and microorganisms, microparticles such as microbeads, etc. through the flow path. Has been. A flow cytometer is known as a typical example of a known particle analyzer. Conventionally, as a flow cytometer, there is one that irradiates a particle flowing through a flow path with a plurality of lights (for example, see Patent Document 1).

Japanese Patent Laid-Open No. 61-283848

In a particle analyzer represented by a flow cytometer, it is desirable that a voltage signal acquired based on an optical signal emitted from a particle irradiated with light contains as little noise as possible.
In contrast, in a flow cytometer that irradiates particles with a plurality of light, a second irradiation unit that irradiates second light downstream of the flow path is provided for the first irradiation unit that irradiates the first light. It has been. In such a conventional flow cytometer, since the acquisition position based on the time axis of the second voltage signal acquired from the second irradiation unit is fixed, the particles flowing through the flow path The second voltage signal is caused by a change in flow velocity, an adjustment error, and other factors that are larger than the acquisition position based on the time axis of the second voltage signal acquired from the second irradiation unit. There is a possibility that the second voltage signal protrudes from the acquisition position based on the time axis, and the second voltage signal is lost.

  A main object of the present technology is to provide a particle analyzing apparatus and a particle analyzing method capable of determining an acquisition position based on a time axis of the second voltage signal.

  As a result of intensive studies to achieve the above object, the inventors of the present application have determined that the first voltage signal output based on the first light irradiation and the flow velocity of the particles are The present inventors have newly found that it is possible to determine the acquisition position based on the time axis of the second voltage signal acquired based on the irradiation of the second light, and have completed the present technology.

That is, the particle analyzer according to the present technology includes a first irradiation unit that irradiates particles flowing through the flow path at a first position on the flow path, and a first irradiation unit that is downstream of the first position. A second irradiation unit that irradiates the particle with second light at a second position; a light detection unit that obtains an optical signal emitted from the particle; and outputs a voltage signal; and the first irradiation unit performs the first irradiation. Based on the first voltage signal output based on the irradiation of one light and the flow velocity of the particles, the first output from the light detection unit based on the irradiation of the second light by the second irradiation unit A signal processing unit that determines an acquisition position based on a time axis of the second voltage signal.
Alternatively, the signal processing unit is configured to determine an acquisition position based on a time axis of the second voltage signal based on a time when the first voltage signal exceeds a threshold and a flow velocity of the particles. May be.
Or the structure which determines the acquisition position based on the time-axis of said 2nd voltage signal based on the flow fluctuation | variation based on a temperature change further may be sufficient as the said signal processing part.
Or the structure which determines the acquisition position based on the time-axis of said 2nd voltage signal based on the flow fluctuation | variation based on the flow velocity adjustment error further may be sufficient as the said signal processing part.
Alternatively, the signal processing unit is configured to output a first voltage signal output from the light detection unit based on a value defined based on the particle size and a time when the first voltage signal exceeds a threshold value. The acquisition position based on the time axis may be determined.
Or the structure which determines the acquisition position based on the time-axis of said 2nd voltage signal based on the value defined based on the magnitude | size of the said particle | grain further may be sufficient as the said signal processing part.
Alternatively, the acquisition position based on the time axis of the first voltage signal and the second voltage signal determined by the signal acquisition unit may include an acquisition start time and an acquisition time range.
The particle analysis method according to the present technology includes a first irradiation step of irradiating a first light to particles flowing through the flow path at a first position on the flow path, and a second irradiation downstream of the first position. A second irradiation step of irradiating the particle with the second light at a position; a light detection step of acquiring a light signal emitted from the particle and outputting a voltage signal; and the first irradiation step of the first irradiation step. Based on the first voltage signal output based on the light irradiation and the flow velocity of the particles, the second output in the light detection step based on the second light irradiation in the second irradiation step. And a signal processing step of determining an acquisition position based on a time axis of the voltage signal.

  According to the present technology, the acquisition position based on the time axis of the second voltage signal can be determined. In addition, the effect described in this specification is an illustration to the last, and is not limited, Moreover, there may exist another effect.

It is a figure showing typically the example of composition concerning the particle analysis device of a 1st embodiment of this art. It is a time chart which shows the example of signal processing concerning the particle analysis device of a 1st embodiment of this art. 3 is a flowchart of a particle analysis method according to the present technology.

Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings. The embodiment described below shows an example of a typical embodiment of the present technology, and the scope of the present technology is not interpreted narrowly. The description of the embodiment will be made in the following order.
1. Particle analyzer (1) Light irradiation unit 11
(2) Photodetector 12
(3) Signal processor 13
(4) Particle 2
(5) Flow path 3
(6) Operation example (7) Signal processing example (8) Offset calculation method Particle analysis method

<1. Particle Analyzer 1>
FIG. 1 is a schematic conceptual diagram schematically showing an embodiment of a particle analyzer 1 according to the present technology. The particle analysis device 1 according to the present technology is a device that analyzes the characteristics of the particles 2 flowing through the flow path 3, and includes at least a light irradiation unit 11, a light detection unit 12, and a signal processing unit 13. Each part will be described below.

(1) Light irradiation unit 11
As shown in FIG. 1, the light irradiation unit 11 irradiates a plurality of light beams LD1 and LD2 corresponding to the positions P1 and P2, respectively, to different positions P1 and P2 of the flow path 3 through which the particles 2 flow. To do. The positions P1 and P2 are displaced from each other in the flow direction of the particles 2.
The number of lights irradiated by the light irradiation unit 11 is not limited to two as shown in FIG. Hereinafter, the nth position (n is 1 to the total number of lights) counted from the upstream side in the flow direction is defined as the nth position, and the light irradiated to the nth position is defined as the nth light. To do.

The light irradiation unit 11 includes a first irradiation unit 11a that irradiates the first light LD1 and a second irradiation unit 11b that irradiates the second light LD2. Note that the light sources of the first irradiation unit 11a and the second irradiation unit 11b are not limited, and may be, for example, a semiconductor laser, that is, a laser diode, a solid laser, or a gas laser. Among these, by using a semiconductor laser, the apparatus can be configured to be small and inexpensive.

  Further, the wavelengths of the light beams LD1 and LD2 are not limited, and different wavelengths can be appropriately selected. The first light LD1 is preferably light having a shorter wavelength than the second light LD2, and more preferably. The wavelength of the first light LD1 is 488 nm corresponding to blue light, and the wavelength of the second light LD2 is 638 nm corresponding to red light. The first and second positions P1 and P2 may be the respective focal points of the first and second lights LD1 and LD2. In the particle analyzer according to the present embodiment, a description will be given of an apparatus in which blue laser light is irradiated as the first light LD1 and red laser light is irradiated as the second light LD2.

The configuration for displacing the first and second positions P1 and P2 in the flow direction of the particles 2 is not limited. For example, the light sources of the respective light LD1 and LD2 may be arranged at intervals in the flow direction.
Further, the light irradiation unit 11 may include a position adjustment structure that adjusts the positions P1 and P2 of the respective lights LD1 and LD2. The position adjustment structure may include a focus adjustment optical system such as a lens or a prism.

(2) Photodetector 12
The light detection unit 12 is configured to acquire a voltage signal based on the fluorescence FL (LD1) and FL (LD2) emitted from the particle 2 by irradiation with the laser beams LD1 and LD2. Although the aspect of the light detection part 12 is not limited, as an example of a preferable aspect, as shown in FIG. 1, the light detection part 12 may include a light separation element 121, a fluorescence detection part 122, and a scattered light detection part 123. . Furthermore, the light detection unit 12 may include a zero-order light removal element 124.

[Light separating element 121]
The light separation element 121 separates the optical signal emitted from the particle 2 by the irradiation of the first laser light LD1 into the fluorescence FL (LD1) and the scattered light S, and the fluorescence FL (LD1) is sent to the fluorescence detection unit 122. Light guide. The light separation element 121 may be configured to transmit the fluorescent light FL (LD1) and reflect the scattered light S. In the particle analyzer 1 according to the present embodiment, the optical signal emitted from the particle 2 by the irradiation of the second laser beam LD2 is not separated by the light separation element 121, but as the fluorescence FL (LD2), the fluorescence detection unit 122. To guide the light. The light separation element 121 may be configured to separate the fluorescence FL (LD2) emitted from the particles 2 by the irradiation of the second laser light LD2 from the scattered light. A plurality of the light separation elements 121 may be arranged so as to correspond to the respective laser beams LD1 and LD2.

  The light separation element 121 may be arranged so as to be inclined with respect to the incident direction of the optical signal from the particle 2. The light separation element 121 may be a wavelength selective mirror that transmits light having a wavelength equal to or greater than a specific wavelength and reflects light having a wavelength less than the specific wavelength. The mirror may be a dichroic mirror or the like.

[Fluorescence detection unit 122]
The fluorescence detection unit 122 is disposed on the traveling direction side of the fluorescence FL (LD1) and FL (LD2) with respect to the light separation element 121. Fluorescence FL (LD1) and FL (LD2) are incident on the fluorescence detection unit 122. The fluorescence detection unit 122 converts the incident fluorescence FL (LD1) and FL (LD2) into voltage signals and outputs them to the signal processing unit 13. The fluorescence detection unit 122 may be a PMT (Photomultiplier Tube) or the like.

[Scattered light detection unit 123]
As shown in FIG. 1, the particle analyzer 1 of the present disclosure includes a scattered light detection unit 123 at a position on the traveling direction side of the scattered light S with respect to the light separation element 121. The scattered light S separated by the light separation element 121 is incident on the scattered light detection unit 123. The scattered light detection unit 123 converts the incident scattered light S into a voltage signal and outputs the voltage signal to the signal processing unit 13. The scattered light detection unit 123 may be a photo detector or the like.

[0th-order light removal element 124]
The 0th-order light removal element 124 blocks the 0th-order light ZL1 and ZL2 such as the laser beams LD1 and LD2 that have traveled straight without being scattered. The zero-order light removal element 124 may be a mask or an optical filter that selectively blocks specific light, but is not limited thereto. The arrangement position of the zero-order light removal element 124 is not limited, and a suitable position in front of the fluorescence detection unit 122 may be selected.

(3) Signal processor 13
The signal processing unit 13 is configured to control the irradiation of the light irradiation unit 11. That is, the signal processing unit 13 is configured to process the voltage signal output from the fluorescence detection unit 122 and the scattered light detection unit 123. The signal processing unit 13 is based on the first voltage signal output by the light detection unit 12 based on the irradiation of the first blue laser light LD1 by the first irradiation unit 11a and the flow velocity of the particles 2. Thus, the acquisition position is determined based on the time axis of the second voltage signal output from the light detection unit 12 based on the irradiation of the second red laser beam LD2 by the second irradiation unit 11b.

  The signal processing unit 13 is configured to determine an acquisition position based on a time axis of the second voltage signal based on a time when the first voltage signal exceeds a threshold and a flow velocity of the particle 2. May be. Further, the signal processing unit 13 may be configured to determine an acquisition position based on the time axis of the second voltage signal based on a flow fluctuation based on a temperature change in the flow path 3. Further, the signal processing unit 13 may be configured to determine an acquisition position based on a time axis of the second voltage signal based on a flow fluctuation based on a flow velocity adjustment error. Further, the signal processing unit 13 outputs the first output from the light detection unit 12 based on a value defined based on the size of the particle 2 and a time when the first voltage signal exceeds a threshold value. The acquisition position based on the time axis of the voltage signal may be determined. Still further, the signal processing unit 13 may be configured to determine an acquisition position based on a time axis of the second voltage signal based on a value defined based on the size of the particle. An acquisition position determination mechanism based on the time axis of the second voltage signal by the signal processing unit 13 will be described later.

In the signal processing unit 13, at the time of initial setting, the flow velocity of the particle 2 according to the present technology, the distance from the position P1 to the position P2 in the flow path 3 (hereinafter, referred to as “spot distance”), A value defined based on the size (hereinafter referred to as “window extension”) is stored. Here, the flow velocity and size of the particle 2 are appropriately determined by the user of the particle analyzer according to the present technology. It is possible to change and set.
Furthermore, the processing in the signal processing unit 13 may include various types of processing for analyzing the characteristics of the particle 2 such as signal quantification, fluorescence correction, and image generation, but is not limited thereto.
The signal processing unit 13 may be configured by an electronic device or the like. The electronic device may include an arithmetic processing device such as a CPU or MPU and a storage device such as a RAM or ROM. The ROM may store a program and data for realizing the function of the signal processing unit 13. The arithmetic processing unit may realize the function of the signal processing unit 13 by executing a program stored in the ROM. The RAM may be used as a work area for the arithmetic processing unit. However, it is not limited to such a configuration.

(4) Particle 2
Particles 2 to be analyzed by the particle analyzer 1 according to the present technology include bio-related microparticles such as cells, microorganisms, and ribosomes, synthetic particles such as latex particles, gel particles, and industrial particles, and initial settings of the particle analyzer 1. Sample particles used in the above may be widely included.

The living body-related microparticles may include chromosomes, ribosomes, mitochondria, and organelles, that is, organelles that constitute various cells. The cells may include plant cells, animal cells, blood cells, and the like. Furthermore, microorganisms may include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and bacteria such as yeast.
The biologically relevant microparticles may also include biologically relevant polymers such as nucleic acids, proteins and complexes thereof.

  The industrial particles may be particles formed of an organic polymer material, an inorganic material, a metal material, or the like. As the organic polymer material, polystyrene, styrene / divinylbenzene, polymethyl methacrylate, or the like may be used. As the inorganic material, glass, silica, a magnetic material, or the like may be used. As the metal material, for example, gold colloid and aluminum may be used. The shape of the microparticles may be spherical or non-spherical, and the size and mass are not particularly limited.

  Further, the particles 2 applicable to the present technology are modified with a fluorescent substance such as a fluorescent dye, a radioactive substance, an intercalator, or a labeling substance such as microbeads so that the light detection unit 12 can detect a signal. It is preferable. For example, when a fluorescent dye is used, the type thereof is not particularly limited, and any known fluorescent dye can be used.

(5) Flow path 3
The flow path 3 is provided in the microchip 30. A sample liquid containing particles 2 is introduced into the flow path 3. The microchip 30 may be formed of various plastics such as glass, polypropylene, polycarbonate, cycloolefin polymer, and polydimethylsiloxane. The material of the microchip 30 is not particularly limited, but as an example of a preferred embodiment, a material having translucency and a small optical error may be employed. The flow path 3 may be provided in advance in the particle analysis apparatus 1 according to the present technology, but a commercially available flow path or the like may be installed in the particle analysis apparatus for analysis.

  The form of the flow path 3 that can be used in the particle analyzer 1 according to the present technology is not particularly limited, and can be freely designed. For example, those formed in a known two-dimensional or three-dimensional plastic or glass substrate T, or those used in conventional flow cytometers may be used in the particle analyzer 1 according to the present technology. Can do.

  Further, the channel width, channel depth, and channel cross-sectional shape of the channel 3 are not particularly limited, and can be freely designed. For example, a microchannel having a channel width of 1 mm or less can also be used in the particle analyzer according to the present technology.

(6) Example of Operation of Particle Analyzer An example of operation of the particle analyzer 1 according to the present technology will be described.

  First, when the particle 2 flowing through the flow path 3 reaches the first position P1, the blue laser light LD1 is irradiated to the particle 2 by the first irradiation unit 11a. Thus, the optical signal emitted from the particle 2 is separated into scattered light S and fluorescence FL (LD1) by the light separation element 121. The scattered light S is incident on the scattered light detection unit 123, and the scattered light detection unit 123 converts the scattered light S into a voltage signal and outputs the voltage signal to the signal processing unit 13.

  Next, when the particle 2 passes through the first position P1 and reaches the second position P2, the second irradiation unit 11b irradiates the particle 2 with the red laser light LD2. As a result, the optical signal generated from the particle 2 is detected as fluorescence FL (LD2) by the fluorescence detection unit 122 and converted into a voltage signal.

  As described above, in the particle analyzer irradiated with two or more lights, the acquisition start time and the acquisition time range of the voltage signal based on the second red laser beam LD2 (hereinafter referred to as “based on the second red laser beam LD2”). If the “acquisition position based on the time axis of the voltage signal” is fixed, the particle diameter of the particles 2 flowing through the flow path 3 is on the time axis of the voltage signal based on the second red laser light LD2. If it is larger than the acquisition position based on it, there is a risk of loss of the voltage signal acquired by the second red laser beam LD2. Alternatively, when the particle diameter of the particles 2 flowing through the flow path 3 is smaller than the voltage signal acquisition time range based on the second red laser light LD2, noise is added to the voltage signal based on the second red laser light LD2. There is a risk of causing an unnecessary increase.

  Therefore, in the particle analysis device 1 according to the present technology, the signal processing unit 13 mainly acquires the acquisition position based on the time axis of the voltage signal based on the second red laser light LD2 irradiated by the second irradiation unit 11b. A decision is made.

(7) Signal Processing Example of Particle Analyzer An example of signal processing by the signal processing unit 13 will be described below with reference to FIG. FIG. 2 is a time chart showing an example of signal processing by the signal processing unit 13.
First, the detection target particle 2 that emits fluorescence is caused to flow based on at least one of the first blue laser beam LD1 and the second red laser beam LD2.
As a result, voltage signals 40 and 50 based on the first blue laser beam LD1 and the second red laser beam LD2 are output to the signal processing unit 13. As a result, the time 41 (hereinafter referred to as “cross-up”) when the voltage signal 40 based on the first blue laser beam LD1 exceeds the threshold 60 is stored in the signal processing unit 13.

  At the same time, the acquisition start time 43 of the voltage signal 40 based on the first blue laser light LD1 is determined by the pre-window extension 42 defined based on the size of the particle 2. Further, the time 44 (hereinafter referred to as “trigger section 44”) during which the voltage signal 40 is output with the threshold 60 or more is stored in the signal processing unit 13. As a result, the acquisition time range 46 of the voltage signal 40 is determined based on the trigger period 44, the pre-window extension 42, and the post-window extension 45 defined based on the size of the particle 2. Here, the pre-window extension 42 and the post-window extension 45 are values defined based on the size of the particles 2 and can be appropriately changed. As shown in FIG. 2, the pre-window extension 42 indicates the time from the start time of the acquisition time range 46 of the voltage signal 40 based on the first blue laser light LD1 to the start time of the trigger section 44. Further, as shown in FIG. 2, the post window extension 45 is detected from the end time of the trigger section 44, that is, from the time when the voltage signal 40 acquired based on the first blue laser light LD 1 divides the threshold 60. The time until the end time of the acquisition time range 46 of the voltage signal 40 is indicated.

  Further, the signal processing unit 13 detects a value 48 (hereinafter referred to as “interval 48”) obtained by dividing the distance between spots stored in advance in the signal processing unit 13 by the flow velocity of the particles 2. Thereby, the signal processing unit 13 determines the time 51 during which the voltage signal 50 based on the second red laser light LD2 exceeds the threshold 60 based on the cross-up 41 and the interval 48.

Further, based on the interval 48, the pre-offset 52 calculated based on the flow velocity of the particles 2, and the pre-window extension 53 by the signal processing unit 13, the second one in accordance with the following formula (1): The acquisition start time 54 of the voltage signal 50 based on the light LD2 is determined.

Formula (1) Cross-up 41 + interval 48 − pre-offset 52 − pre-window extension 53

  Here, the pre-offset 52 according to the equation (1) is a value calculated based on the flow velocity of the particles 2, and in order to obtain the voltage signal 50 based on the second red laser beam LD2 without omission. This is a value (margin) to be provided. The pre-offset 52 indicates the time from the acquisition start time 54 of the voltage signal 50 based on the second red laser beam LD2 to the start time of the pre-window extension 53, as shown in FIG. The pre-window extension 53 according to the formula (1) is a value calculated based on the size of the particle 2 and can be changed as appropriate. As shown in FIG. 2, the pre-window extension 53 indicates the time from the end time of the pre-offset 52 to the time when the voltage signal 50 acquired from the second red laser beam LD2 reaches the threshold 60. .

The signal processing unit 13 calculates the pre-offset 52 and the post-offset 56 calculated based on the flow velocity of the particles 2, the pre-window extension 53 and the post-window extension 57 recorded in advance in the signal processing unit 13, and the trigger section 44. Then, the acquisition time range 58 of the voltage signal 50 acquired based on the second red laser beam LD2 is determined.
Specifically, the acquisition time range 58 of the voltage signal 50 acquired based on the second red laser beam LD2 is determined in accordance with the following equation (2).

Formula (2) Pre-offset 52 + Pre-window extension 53 + Trigger section 44 + Post-window extension 57 + Post-offset 56

Here, the post window extension 57 according to the expression (2) is a value calculated based on the size of the particle 2 and can be changed as appropriate. As shown in FIG. 2, the post window extension 57 sets the time from the time when the voltage signal 50 acquired based on the second red laser beam LD2 divides the threshold 60 to the start time of the post offset 56. Point to.
Further, the post-offset 56 according to the above formula is a value calculated based on the flow velocity of the particle 2, and is a value provided for acquiring the voltage signal 50 based on the second red laser light LD2 without omission. . As shown in FIG. 2, the post offset 56 indicates the time from the end time of the post window extension 57 to the end time of the acquisition time range 58 of the voltage signal 50 based on the second red laser beam LD2.

  In the particle analyzer 1 according to the present technology, the signal processing unit 13 determines the acquisition start time 54 of the voltage signal 50 based on the second red laser beam LD2 and the acquisition time range 58 of the voltage signal 50. Although it is a structure, these determinations can be made by using the first voltage signal based on the scattered light detected by the scattered light detection unit 123 as a trigger. Or it can also be performed by using the first voltage signal based on the fluorescence detected by the fluorescence detector 122 as a trigger.

As described above, in the particle analyzer 1 according to the present technology, the signal processing unit 13 causes the acquisition start time 54 of the voltage signal 50 based on the second red laser light LD2, the acquisition time range 58 of the voltage signal 50, and However, the flow rate of the particles 2 fluctuates according to the temperature change of the flow path 3.
Here, the pre-offset 52 and the post-offset 56 that determine the acquisition time range 58 of the voltage signal 50 based on the second red laser beam LD2 are calculated based on the flow velocity of the particles 2. That is, the pre-offset 52 and the post-offset 56 vary according to the temperature change of the flow path 3.
When the pre-offset 52 and the post-offset 56 are set to values larger than necessary, the acquisition time range 58 of the voltage signal 50 based on the second red laser light LD2 is also set to a large value. As a result, for example, noise due to the laser light may unnecessarily increase in the voltage signal 50 based on the second red laser light LD2.
For this reason, the values of the pre-offset 52 and the post-offset 56 are preferably as small as possible. The following method can be considered as a method of calculating the offset. Note that the following method is merely an example of a method for obtaining an offset, and the method for obtaining an offset is not limited to these.

(8) Offset calculation method [First method: Method using flow velocity adjustment center value]
(First step) The flow rate of the sample particles is adjusted by allowing the sample particles that emit fluorescence based on both the first blue laser light LD1 and the second red laser light LD2 to flow through the flow path 3. .
At this time, an adjustment error is taken in a range of ± 5% with respect to the flow velocity of the sample particles, and a flow velocity adjustment center value is obtained.
(Second step) The interval is calculated based on the flow velocity adjustment center value (flow velocity) calculated in the first step and the inter-spot distance stored in the signal processing unit 13.
Then, the offset is calculated in consideration of the flow velocity adjustment error in the first process stored in the signal processing unit 13 in advance and the flow fluctuation due to the temperature change.

Hereinafter, how to obtain the offset by the first method using real values will be described.
That is, when the flow rate of the sample particles in the first step is 5 m / s, the range of 4.75 to 5.25 m / s is set as the adjustment error range.
Furthermore, when the distance between spots is 80 μm, the interval is 16 us (80 μm / 5 m / s).
Then, the flow fluctuation due to the temperature change is set to ± 1.5% flow rate fluctuation width, and further, ± 2 to prevent noise from occurring in the voltage signal based on the second red laser beam LD2 as much as possible. When a margin of 0.0% is secured, an offset of 1.36 us (16 * (5 + 1.5 + 2) / 100) is calculated as a result of considering the flow rate adjustment error, the fluctuation width of the flow fluctuation due to the temperature change, and the margin. .

[Second method: Method using measured value at flow rate adjustment]
(First Step) Sample particles that emit fluorescence based on both the first blue laser light LD1 and the second red laser light LD2 are passed through the flow path 3, and the flow velocity of the sample particles is measured. .
(Second step) The interval is calculated from the value of the flow velocity measured in the first step.
Then, the offset is calculated in consideration of the flow fluctuation due to the temperature change.
This second method will be described using real values.
Similar to the first method, the interval is 16 us.
Furthermore, in the second method, since the measurement value in the first step is used, it is not necessary to consider the flow rate adjustment error. Therefore, the offset can be calculated in consideration of only the fluctuation width and margin of the flow fluctuation due to the temperature change, and the offset is 0.57 us (16 * (1.5 + 2) / 100).

[Third method: Method using measured value and temperature change during flow rate adjustment]
Since the first step and the second step in the third method are the same as the first step and the second step of the second method, the description thereof is omitted here.
Moreover, in the particle analyzer according to the present technology, the flow fluctuation due to the temperature change can be suppressed as much as possible by making the water pressure in the flow path 3 constant by an external device or the like.
Furthermore, in this third method, the flow rate of the sample particles is measured as needed, and the interval is detected based on this flow rate.
For this reason, in the third method, the offset can be calculated in consideration of only the margin.
That is, when a margin of ± 2.0% is secured, the offset is 0.32 us (16 * 2/100).

  As described above, in the particle analyzer 1 of the present technology, as illustrated in FIG. 2, the signal processing unit 13 causes the acquisition start time 54 of the voltage signal 50 based on the second red laser light LD2 and the voltage to be obtained. The acquisition time range 58 of the signal 50 can be determined, so that the second voltage signal 50 can be acquired with higher accuracy than before. Thereby, the S / N ratio (signal-noise ratio) can be improved, and the analysis accuracy of the particles 2 can be improved accordingly.

  Further, in the particle analyzer 1 of the present technology, the offset value can be reduced by considering not only the flow velocity of the particles 2 but also the flow fluctuation based on the temperature change and the flow velocity adjustment error. As a result, the acquisition time range 58 of the voltage signal 50 based on the second red laser beam LD2 can be reduced within a necessary range, and thus the second voltage signal 50 can be acquired with higher accuracy than before. Is possible. Thereby, the S / N ratio (signal-noise ratio) can be improved. Furthermore, in calculating the offset, by using the actual measured value of the flow velocity of the particles 2, it is possible to calculate the offset mainly based on flow fluctuations due to temperature changes. As a result, the offset can be set to a smaller value, so that the second voltage signal 50 with higher accuracy can be acquired more reliably.

<2. Particle analysis method>
FIG. 3 is a flowchart of the particle analysis method according to the present technology. The particle analysis method according to the present technology is roughly divided into a method of performing at least the first irradiation step I, the second irradiation step II, the light detection step III, and the signal processing step IV. The details of the first irradiation step I, the second irradiation step II, the light detection step III, and the signal processing step IV are as follows: the first irradiation unit 11a, the second irradiation unit 11b of the particle analyzer 1 according to the present technology described above. Since the light detection unit 12 and the signal processing unit 13 are the same as the respective methods, the description is omitted here.

Moreover, this technique can also take the following structures.
(1) a first irradiation unit that irradiates particles that flow through the flow path at a first position on the flow path with a first light; and a second position that is downstream of the first position. A second irradiation unit that irradiates second light, a light detection unit that obtains an optical signal emitted from the particles and outputs a voltage signal, and an output based on the irradiation of the first light by the first irradiation unit On the time axis of the second voltage signal output from the light detection unit based on the irradiation of the second light by the second irradiation unit based on the first voltage signal and the flow velocity of the particles A particle processing apparatus comprising: a signal processing unit that determines an acquisition position based thereon.
(2) The signal processing unit determines an acquisition position based on a time axis of the second voltage signal based on a time when the first voltage signal exceeds a threshold and a flow velocity of the particles (1 ) The particle analyzer described above.
(3) The particle analyzer according to (2), wherein the signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on a flow fluctuation based on a temperature change. (4) The particle analyzer according to (3), wherein the signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on a flow fluctuation based on a flow velocity adjustment error.
(5) The signal processing unit is configured to output a first voltage output from the light detection unit based on a value defined based on a size of the particle and a time when the first voltage signal exceeds a threshold value. The particle analyzer according to (4), wherein an acquisition position based on a time axis of a signal is determined.
(6) The particle analyzer according to (5), wherein the signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on a value defined based on the size of the particle. .
(7) The acquisition position based on the time axis of the first voltage signal and the second voltage signal determined by the signal processing unit includes an acquisition start time and an acquisition time range. Analysis equipment. (8) a first irradiation step of irradiating the particles flowing through the flow path at a first position on the flow path with a first light; and a second position downstream of the first position to the particles at the first position. A second irradiation step of irradiating the second light, a light detection step of acquiring a light signal emitted from the particles and outputting a voltage signal, and an output based on the irradiation of the first light by the first irradiation step On the time axis of the second voltage signal output in the light detection step based on the irradiation of the second light in the second irradiation step based on the first voltage signal and the flow velocity of the particles A particle processing method comprising: a signal processing step of determining a based acquisition position.

11a 1st irradiation part 11b 2nd irradiation part 12 Photodetection part 13 Signal processing part 2 Particle | grain 3 Flow path

Claims (8)

A first irradiation unit that irradiates the first light to particles flowing through the flow path at a first position on the flow path;
A second irradiation unit that irradiates the particles with a second light at a second position downstream of the first position;
A light detection unit that obtains an optical signal emitted from the particles and outputs a voltage signal;
Based on the first voltage signal output based on the irradiation of the first light by the first irradiation unit and the flow velocity of the particles, based on the irradiation of the second light by the second irradiation unit. A signal processing unit for determining an acquisition position based on a time axis of the second voltage signal output from the light detection unit;
Particle analyzer including The signal processing unit determines an acquisition position based on a time axis of the second voltage signal based on a time when the first voltage signal exceeds a threshold and a flow velocity of the particles.
The particle analyzer according to claim 1. The signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on a flow fluctuation based on a temperature change.
The particle analyzer according to claim 2. The signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on flow fluctuation based on a flow velocity adjustment error.
The particle analyzer according to claim 3. The signal processing unit is configured to output the first voltage signal output from the light detection unit based on a value defined based on the size of the particle and a time when the first voltage signal exceeds a threshold value. Determine the acquisition position based on the time axis,
The particle analyzer according to claim 4. The signal processing unit further determines an acquisition position based on a time axis of the second voltage signal based on a value defined based on the particle size.
The particle analyzer according to claim 5. The acquisition position based on the time axis of the first voltage signal and the second voltage signal determined by the signal processing unit includes an acquisition start time and an acquisition time range.
The particle analyzer according to claim 6. A first irradiation step of irradiating a first light to particles flowing through the flow path at a first position on the flow path;
A second irradiation step of irradiating the particles with a second light at a second position downstream of the first position;
A light detection step of acquiring a light signal emitted from the particles and outputting a voltage signal;
Based on the first voltage signal output based on the irradiation of the first light in the first irradiation step and the flow velocity of the particles, based on the irradiation of the second light in the second irradiation step. A signal processing step for determining an acquisition position based on a time axis of the second voltage signal output in the light detection step;
A particle analysis method comprising:

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