WO2020230779A1 - Particle dispensing apparatus, particle dispensing method, and computer program - Google Patents

Abstract

A particle dispensing apparatus (100) is provided with: a laser device (11) for continuously outputting laser light in a deep ultraviolet light region; a detection unit (13) for detecting fluorescence of particles obtained by irradiating the particles with the laser light, in the deep ultraviolet light region, outputted from the laser device (11); and a control circuit (14) for dispensing the particles on the basis of the detected fluorescence.

Description

Particle sorting device, particle sorting method and computer program

The present invention relates to a particle detection technique.
The present application claims priority based on Japanese Patent Application No. 2019-090838 filed in Japan on May 13, 2019, the contents of which are incorporated herein by reference.

Conventionally, flow cytometry is known, which can discriminate various characteristics at high speed by irradiating fine particles flowing in an ultrafine water stream with a laser. Flow cytometry can analyze not only biological cells but also various fine particles. For example, flow cytometry can acquire forward scattered light that reflects the size of particles, side scattered light that reflects the internal structure of particles, and fluorescence generated by shining a laser on the fine particles to be measured at high speed for each particle. Is. A cell sorter is a device that performs feedback control based on the acquired data and has a mechanism for separating specific particles.

Until now, in order to specifically detect biological cells, an operation using a dye that specifically adsorbs DNA (Deoxyribonucleic acid), RNA (Ribonucleic acid), amino acids, etc. existing in the cells (generally). , Called dyeing) has been performed. This dye includes a dye for transmission observation and a fluorescent dye. A dye for transmission observation is a dye that literally appears to be colored and its presence can be identified by a general microscope. A fluorescent dye is a dye that excites a dye molecule by irradiating it with light of a specific wavelength, emits light shifted to a long wavelength side, and its existence can be identified by a fluorescence microscope or the like. In order to obtain higher detection sensitivity, it is common to stain using a fluorescent dye, and cell staining using a fluorescent dye has also been often performed in the staining of microorganisms.

However, in this staining, it is necessary for the dye for staining to permeate into the cells, and there are the following technical problems. For example, in the case of life such as spores, which significantly restricts mass transfer to the outside world, there is a problem that even if biomolecules to be stained are contained in cells, they cannot be stained. The dye for dyeing is composed of the same elements as those constituting biological cells such as carbon, hydrogen, and oxygen. Therefore, the introduction of the dye into the cell changes the elemental composition of the cell itself, which hinders precise elemental analysis.

As a method for solving the problem caused by dyeing, a technique for visualizing particles on an inorganic substrate (gypsum) without dyeing has been proposed (see, for example, Patent Document 1). Patent Document 1 describes a method and an apparatus for measuring fluorescence or Raman light by using a laser or an illumination apparatus in a deep ultraviolet light region of 200 nm to 300 nm. In the technique of Patent Document 1, the laser beam is intermittent (pulse oscillation), and oscillation and stop are repeated about 1 to 20 times per second.

U.S. Pat. No. 7525653

However, when the laser described in Patent Document 1 is used in a cell sorter capable of high-speed processing, particles cannot be detected during the time when the laser oscillation is stopped. Therefore, there is a problem that the detection accuracy of particles is lowered.

In view of the above circumstances, an object of the present invention is to provide a technique capable of improving the detection accuracy of particles.

One aspect of the present invention is a laser device that continuously outputs a laser beam in a deep ultraviolet light region, and the particles obtained by irradiating a particle with a laser beam in the deep ultraviolet light region output from the laser device. It is a particle sorting device including a detection unit for detecting the fluorescence of the above, and a control circuit for sorting the particles based on the detected fluorescence.

One aspect of the present invention is the above-mentioned particle sorting device, which is a region in the deep ultraviolet light region where autofluorescence due to an inorganic mineral is not observed.

One aspect of the present invention is the above-mentioned particle sorting device, and the deep ultraviolet light region is a region in the vicinity of 230 nm.

One aspect of the present invention is the above-mentioned particle sorting device, in which the particles are discharged by a jet-in-air method.

One aspect of the present invention detects the fluorescence of the particles obtained by irradiating the particles with the laser light in the deep ultraviolet light region output from the laser device that continuously outputs the laser light in the deep ultraviolet light region. It is a particle sorting method including a detection step and a sorting step of sorting the particles based on the detected fluorescence.

One aspect of the present invention detects the fluorescence of the particles obtained by irradiating the particles with the laser light in the deep ultraviolet light region output from the laser device that continuously outputs the laser light in the deep ultraviolet light region. It is a computer program for causing a computer to perform a detection step and a preparative step of preparating the particles based on the detected fluorescence.

According to the present invention, it is possible to improve the particle detection accuracy.

It is a block diagram of the particle sorting apparatus 100 in this embodiment. It is an internal block diagram of the detection unit 13. It is a figure which shows the internal structure of the 1st POD 136-1 (488 nm). It is a figure which shows the internal structure of the 2nd POD 136-2 (230 nm). It is a figure which shows the internal structure of the 3rd POD 136-3 (355 nm). It is a flowchart which shows the process flow of the particle sorting apparatus 100. It is a figure which shows the detection result of fluorescence obtained by irradiating a spore with deep ultraviolet light. It is a figure which shows the detection result of fluorescence obtained by irradiating a vegetative cell with deep ultraviolet light. It is a figure which shows the micrograph which contains only a vegetative cell. It is a figure which shows the result of having analyzed the state of FIG. 8A by a fluorescence spectrophotometer. It is a figure which shows the micrograph which contains a vegetative cell and a spore. It is a figure which shows the result of having analyzed the state of FIG. 8C by a fluorescence spectrophotometer. It is a figure which shows the micrograph which contains only a spore. It is a figure which shows the result of having analyzed the state of FIG. 8E by a fluorescence spectrophotometer. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result. It is a figure which shows the specific example of the experimental result.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a configuration diagram of a particle sorting device 100 according to the present embodiment. The particle sorting device 100 detects the fluorescence of the particles obtained by irradiating the particles with laser light in the deep ultraviolet light region (hereinafter, simply referred to as “deep ultraviolet light”), and the particles are based on the detected fluorescence of the particles. It is a device that sorts the particles. The target particles in this embodiment are biological cells and spores having DNA, RNA, amino acids and the like. The particles in this embodiment are undyed or unstained particles. The size of the particles is, for example, 0.5 to 20 microns.

The particle sorting device 100 includes a nozzle 10, a plurality of laser devices 11 (11-1 to 11-3), a plurality of focus lenses 12, a detection unit 13, a control circuit 14, a display unit 15, a polarizing plate 16, and a waste liquid tank 17. , A well plate 18 and a photographing device 19. In the following description, the plurality of laser devices 11 will be described as the first laser device 11-1, the second laser device 11-2, and the third laser device 11-3.

The nozzle 10 discharges the sheath liquid 1 and the sample liquid by a jet-in-air method. The sample solution contains particles 2. By the jet-in-air method, the particles 2 are aligned in a row at the center of the flow of the sheath liquid 1. The nozzle 10 is provided with an oscillator, and the oscillator vibrates up and down in a predetermined cycle according to the control of the control circuit 14. As a result, the droplet 3 is formed. The vibration cycle of the vibrator and the pressure in the nozzle 10 are controlled by the control circuit 14.

The first laser device 11-1 is a laser device that continuously outputs laser light in the visible light region (hereinafter, simply referred to as "visible light"). For example, the first laser device 11-1 continuously outputs a laser beam of 488 nm as visible light.
The second laser device 11-2 is a laser device that continuously outputs deep ultraviolet light. For example, the second laser device 11-2 continuously outputs a laser beam of 230 nm as deep ultraviolet light.
The third laser device 11-3 is a laser device that continuously outputs laser light in the ultraviolet light region (hereinafter, simply referred to as "ultraviolet light"). For example, the third laser device 11-3 continuously outputs a laser beam of 355 nm as ultraviolet light.

Here, a method for generating 230 nm laser light will be described. In the present embodiment, the second laser device 11-2 is configured by using a laser unit and a wavelength conversion unit. The laser unit is, for example, a laser device that continuously outputs a laser beam of 460 nm. The wavelength conversion unit is a device that outputs double harmonics. The wavelength conversion unit generates a second harmonic by interacting the laser light output from the laser unit with a nonlinear optical crystal and generating a laser light having a frequency twice that of the laser light output from the laser unit. .. As a result, the 460 nm laser light output from the laser unit is converted into light having twice the frequency. That is, the 460 nm laser light output from the laser unit is converted into the 230 nm laser light having a wavelength of 1/2. Strictly speaking, the laser light after conversion is a laser light in the vicinity of 230 nm (± several nm of 230 nm) in consideration of conversion efficiency and the like.

The focus lenses 12-1 to 12-3 adjust the optical axis of the laser light output from the laser device 11 associated with each other.
The detection unit 13 detects the fluorescence of the particles based on the scattered light obtained by irradiating the particles with the laser light output from the laser device 11 and the fluorescence of the particles (hereinafter referred to as “acquired light”). A specific description will be described later.

The control circuit 14 is based on the fluorescence of particles obtained by the laser light output from the laser device 11 of any of the first laser device 11-1, the second laser device 11-2, and the third laser device 11-3. And separate the particles. The user may decide whether to target the laser light output from the laser device 11 of any of the first laser device 11-1, the second laser device 11-2, and the third laser device 11-3. .. The control circuit 14 applies a positive or negative charge to the droplet 3 containing the particles to be sorted. As a method of applying an electric charge, a method generally used in a cell sorter can be applied. In the following description, the control circuit 14 will be described as separating particles based on the fluorescence of the particles obtained by the laser light output from the second laser device 11-2 that continuously outputs deep ultraviolet light. ..

The number of target laser devices 11 may be plural. The control circuit 14 causes the display unit 15 to display the detection result of the detection unit 13. The control circuit 14 controls the nozzle 10 based on an image captured by the photographing device 19 (hereinafter referred to as “photographed image”). For example, the control circuit 14 changes the pressure in the nozzle 10 when a change of the droplet 3 or more occurs in the captured image. Further, the control circuit 14 changes the vibration cycle of the vibrator when the droplet 3 changes by a threshold value or more.

The display unit 15 is an image display device such as a liquid crystal display or an organic EL (Electro Luminescence) display. The display unit 15 displays the detection result and the captured image. The display unit 15 may be an interface for connecting the image display device to the particle sorting device 100. In this case, the display unit 15 generates a video signal for displaying the detection result and the captured video, and outputs the video signal to the image display device connected to the display unit 15.
The polarizing plate 16 is composed of a first plate having a positive voltage and a second plate having a negative voltage. The first plate and the second plate are installed so as to face each other. A predetermined potential difference (for example, ± 4000 V) is generated between the first plate and the second plate. The charged droplet 3 is separated by the polarizing plate 16.

The waste liquid tank 17 is a tank for discarding the unseparated droplets 3.
The well plate 18 is a flat plate having a plurality of recesses. The well plate 18 collects the separated droplets 3. The well plate 18 can be moved up, down, left and right according to the control of the control circuit 14.
The photographing device 19 is a photographing device such as a camera. The photographing device 19 is fixedly installed at a position where the droplet 3 is formed, and photographs the droplet 3. The photographing device 19 outputs the photographed image of the droplet 3 to the control circuit 14.

FIG. 2 is an internal configuration diagram of the detection unit 13.
As shown in FIG. 2, the detection unit 13 includes an objective lens 131, a tube lens 132, a pinhole plate 133, a prism 134, a plurality of collimation lenses 135-1 to 135-3, a first POD 136-1, a second POD 136-2, and the like. A third POD 136-3 is provided.
The objective lens 131 collects the incident light. Laser light and acquired light are incident on the objective lens 131.
The tube lens 132 refocuses the light output from the objective lens 131.
The pinhole plate 133 is a plate in which pinholes are opened for several minutes of the laser device 11. The pinhole plate 133 blocks unnecessary light from the light obtained from each laser device 11.

The prism 134 is a polyhedron that reflects the light output through the pinhole plate 133. For example, the prism 134 reflects the laser light and the acquired light corresponding to the first laser device 11-1 to the first POD 136-1. For example, the prism 134 reflects the laser light and the acquired light corresponding to the second laser device 11-2 to the second POD 136-2. For example, the prism 134 reflects the laser light and the acquired light corresponding to the third laser device 11-3 to the third POD 136-3.
The collimation lenses 135-1 to 135-3 convert the incoming light into parallel light.

The first POD 136-1 inputs the laser light and the acquired light corresponding to the first laser device 11-1. The first POD136-1 detects the fluorescence of the particles from the input acquired light. The laser light (visible light) corresponding to the first laser device 11-1 is blocked at the first POD 136-1.
The second POD 136-2 inputs the laser light and the acquired light corresponding to the second laser device 11-2. The second POD 136-2 detects the fluorescence of the particles from the input acquired light. The laser light (deep ultraviolet light) corresponding to the second laser apparatus 11-2 is blocked at the second POD 136-2.
The third POD 136-3 inputs the laser light and the acquired light corresponding to the third laser device 11-3. The third POD 136-3 detects the fluorescence of the particles from the input acquired light. The laser light (ultraviolet light) corresponding to the third laser apparatus 11-3 is blocked at the third POD 136-3.

Next, the configuration of each POD will be described with reference to FIGS. 3 to 5.
FIG. 3 is a diagram showing the internal configuration of the first POD 136-1 (488 nm).
As shown in FIG. 3, the first POD 136-1 includes a plurality of dichroic mirrors 137 to 142 and a plurality of absorption filters 143 to 149. Although not shown, a photomultiplier tube (PMT) is provided at the output destination of each absorption filter 143 to 149.

The dichroic mirrors 137 to 142 reflect light having a specific wavelength and transmit light other than the specific wavelength.
The absorption filters 143 to 149 allow light of a specific wavelength to pass through and absorb light other than the specific wavelength. For example, the absorption filter 143 transmits light having a half width of 62 nm centered on 785 nm.
The photomultiplier tube detects the light that has passed through the absorption filters 143 to 149. The photomultiplier tube then amplifies the detected light. Then, the photomultiplier tube converts the amplified optical signal into an electric signal. The photomultiplier tube outputs an electric signal to the control circuit 14.

FIG. 4 is a diagram showing the internal configuration of the second POD 136-2 (230 nm).
As shown in FIG. 4, the second POD 136-2 includes a plurality of dichroic mirrors 150 to 152 and a plurality of absorption filters 153 to 156. Although not shown, a photomultiplier tube is provided at the output destination of each absorption filter 153 to 156.
The dichroic mirrors 150 to 152 reflect light having a specific wavelength and transmit light other than the specific wavelength.
The absorption filters 153 to 156 allow light of a specific wavelength to pass through and absorb light other than the specific wavelength. For example, the absorption filter 153 transmits light having a half width of 36 nm centered on 272 nm.
The photomultiplier tube detects the light that has passed through the absorption filters 153 to 156. The photomultiplier tube then amplifies the detected light. Then, the photomultiplier tube converts the amplified optical signal into an electric signal. The photomultiplier tube outputs an electric signal to the control circuit 14.

FIG. 5 is a diagram showing the internal configuration of the third POD 136-3 (355 nm).
As shown in FIG. 5, the third POD 136-3 includes a plurality of dichroic mirrors 157 to 159 and a plurality of absorption filters 160 to 163. Although not shown, a photomultiplier tube is provided at the output destination of each absorption filter 160 to 163.
The dichroic mirrors 157 to 159 reflect light having a specific wavelength and transmit light other than the specific wavelength.
The absorption filters 160 to 163 allow light of a specific wavelength to pass through and absorb light other than the specific wavelength. For example, the absorption filter 160 transmits light having a half width of 50 nm centered on 457 nm.
The photomultiplier tube detects the light that has passed through the absorption filters 160-163. The photomultiplier tube then amplifies the detected light. Then, the photomultiplier tube converts the amplified optical signal into an electric signal. The photomultiplier tube outputs an electric signal to the control circuit 14.

FIG. 6 is a flowchart showing a processing flow of the particle sorting device 100. At the start of the process of FIG. 6, it is assumed that the particles are discharged by the jet-in-air method. In FIG. 6, the processing flow will be described with a focus on the second laser apparatus 11-2.
The second laser device 11-2 irradiates the laser light (deep ultraviolet light) (step S101). When the particles are irradiated with the laser light, the acquired light is input to the detection unit 13. On the other hand, when the laser light is not applied to the particles, the laser light is input to the detection unit 13. The input light is input to the second POD 136-2 in the detection unit 13. The second POD 136-2 classifies the input light into each wavelength (step S102). Specifically, the second POD 136-2 classifies the input light into each wavelength by the dichroic mirrors 150 to 152 and the absorption filters 153 to 156. The classified light is input to the photomultiplier tube.

The control circuit 14 determines whether or not fluorescence has been detected (step S103). The determination as to whether or not fluorescence is detected is performed based on whether or not an electric signal exceeding the Thrashold value set in the control circuit 14 is input. That is, whether or not fluorescence is detected is determined by whether or not the intensity of the light input to the photomultiplier tube exceeds a certain value. When light having a light intensity exceeding a certain value is input to the photomultiplier tube, an electric signal exceeding the Thrashold value is input to the control circuit 14. On the other hand, when the light whose intensity exceeds a certain value is not input to the photomultiplier tube, the electric signal exceeding the Thrashold value is not input to the control circuit 14.

When an electric signal exceeding the Throld value is input to the control circuit 14, it is determined that the control circuit 14 has detected fluorescence. On the other hand, when no electric signal exceeding the Throld value is input to the control circuit 14, it is determined that the control circuit 14 has not detected fluorescence. The Treshold value depends on the voltage applied to the photomultiplier tube.

When fluorescence is detected (step S103-YES), the control circuit 14 determines whether or not the source of fluorescence is particles to be selected (for example, vegetative cells, spores) based on the input electrical signal (step S103-YES). Step S104). Specifically, the control circuit 14 determines that the source of fluorescence is the particles to be selected when the characteristics based on the electric signal indicate the characteristics of the particles to be selected. On the other hand, the control circuit 14 determines that the source of fluorescence is not the particles to be selected when the characteristics based on the electric signal do not show the characteristics of the particles to be selected.

Spectral characteristics can be mentioned as characteristics based on electrical signals. Here, in deep ultraviolet light near 230 nm, the peak of the fluorescence spectrum of the vegetative cell appears near 350 nm, and the peak of the fluorescence spectrum of the spore appears near 300 nm. Therefore, the control circuit 14 determines that the source of fluorescence is the particle to be selected when the characteristic based on the electric signal matches any of the characteristics of the above particles. On the other hand, when the characteristic based on the electric signal does not match any of the characteristics of the above particles, the control circuit 14 determines that the source of fluorescence is not the particle to be selected.

When the source of fluorescence is the particles to be sorted (step S104-YES), the control circuit 14 applies an electric charge to the particles to be sorted (step S105). Specifically, the control circuit 14 applies an electric charge to the target particles at the timing when the droplet 3 containing the particles to be sorted is formed. The control circuit 14 may apply a positive charge in the case of vegetative cells and a negative charge in the case of spores. As a result, the charged droplet 3 is deflected by the polarizing plate 16 and collected by the well plate 18.

After that, the control circuit 14 determines whether or not the processing is completed (step S106). When the process is completed (step S106-YES), the particle sorting device 100 ends the process of FIG.
On the other hand, when the processing is not completed (step S106-NO), the particle sorting device 100 executes the processing after step S102.
When fluorescence is not detected in the process of step S103 (step S103-NO), or when the source of fluorescence is not the particles to be sorted in the process of step S104 (step S104-NO), the particle sorting device 100 Executes the process of step S106.

Next, the detection result of fluorescence obtained by irradiating deep ultraviolet light will be described with reference to FIGS. 7A and 7B. In FIGS. 7A and 7B, the fluorescence detected by the photomultiplier tube provided at the output destination of the absorption filter 155 and the fluorescence detected by the photomultiplier tube provided at the output destination of the absorption filter 156 are plotted in two dimensions. It is shown how it was done.
FIG. 7A is a diagram showing the detection result of fluorescence obtained by irradiating spores with deep ultraviolet light. In FIG. 7A, fluorescence of spores is detected in the region indicated by F. FIG. 7B is a diagram showing the detection result of fluorescence obtained by irradiating vegetative cells with deep ultraviolet light. In FIG. 7B, fluorescence of vegetative cells is detected in the region indicated by E.

Next, the photomicrograph and the analysis result by the fluorescence spectrophotometer will be described with reference to FIGS. 8A to 8F.
FIG. 8A is a diagram showing a micrograph containing only vegetative cells 170. FIG. 8B is a diagram showing the result of analyzing the state of FIG. 8A by a fluorescence spectrophotometer. FIG. 8C is a diagram showing a micrograph containing vegetative cells 170 and spores 171. FIG. 8D is a diagram showing the result of analyzing the state of FIG. 8C by a fluorescence spectrophotometer. FIG. 8E is a diagram showing a micrograph containing only spores 171. FIG. 8F is a diagram showing the result of analyzing the state of FIG. 8E by a fluorescence spectrophotometer.
Comparing FIG. 8B and FIG. 8F, the feature of spore 171 alone appears in the vicinity of 300 nm as compared with that of vegetative cell 170 alone.

According to the particle sorting device 100 configured as described above, it is possible to improve the particle detection accuracy. Hereinafter, this effect will be described in detail.
The particle sorting device 100 sorts particles based on the fluorescence of the particles obtained by irradiating the particles with the laser light output from the laser device 11 that continuously outputs deep ultraviolet light. Here, the particle sorting device 100 in the present embodiment uses deep ultraviolet light in which autofluorescence due to minerals is not observed and the particles naturally fluoresce. This makes it possible to detect cells that could not be detected in the past (for example, spores that cannot be found by staining and cells that cannot be stained). Therefore, it is possible to improve the particle detection accuracy.

Conventionally, particles cannot be detected while the laser oscillation is stopped. Therefore, a large number of particles may be dropped. On the other hand, in the particle sorting device 100 of the present invention, a laser device 11 that continuously outputs laser light is used. Therefore, the number of particles can be reduced as compared with the conventional case. Therefore, it is possible to improve the particle detection accuracy. The particle sorting device 100 detects the fluorescence of the particles obtained by irradiating the particles with laser light, and sorts the particles based on the detected fluorescence. As a result, it becomes possible to improve the accuracy of particle sorting.

In the particle sorting apparatus 100, undyed particles are used as the sorting target. Therefore, the elemental composition of the cell itself is not changed. Therefore, more accurate elemental analysis becomes possible.
Examples of the emission method of the particles to be sorted include a jet-in-air method and a flow cell method. Among these emission methods, in the flow cell method, the laser light in the deep ultraviolet light region cannot be used because the laser light in the deep ultraviolet light region may not pass depending on the material used. On the other hand, in the jet-in-air method, laser light in the deep ultraviolet region can be used. Therefore, the particle sorting device 100 in the present embodiment discharges the particles to be sorted by the jet-in-air method. Therefore, analysis can be performed using laser light in the deep ultraviolet light region.

Hereinafter, the experimental results using the particle sorting device 100 will be described.
First, as an outline of the experiment, the experimental method will be described.
<Experimental sample>
Two types of culture medium of Bacillus megaterium (vegetative, spore)
<Liquid medium used>
vegetative: NBRC No.702, spore: DSM (Difco Sporulation Medium)
<Laser conditions>
Deep UV (deep ultraviolet light), 488 nm (visible light: blue laser) was used. The laser device 11 was left ON during sorting.
<Number of sorted cells (number of events)>
1, 2, 5, 10
<Sorting conditions (total)>
Laser (1 condition) x number of sorted cells (4 conditions) = 4 conditions (96-well plate: 1 condition, 48 wells used)
<Culture conditions after sorting>
vegetative: 30 ° C, over night, spore: 37 ° C, over night

<Preparation of sample solution>
Culture of Bacillus megaterium (vegetative cells) -Overnight culture (30 ° C, 100 rpm) by adding glycerol stock (strain) to NBRC702 liquid medium.
-The next day, it will start to become cloudy in about 30 μl in about 3 hours with respect to about 7 mL of medium to be subplanted in a new medium.
-Stop the culture at the stage of turbidity and place it on ice.-When sorting, flow the culture solution directly.
Culture of Bacillus megaterium (spores) -Culturing for 52 hours (37 ° C, 300 rpm) by adding glycerol stock (strain) to DSM liquid medium.
-When sorting, the solution diluted with DSM liquid medium is poured directly.
<Dyeing conditions>
It is unstained and flows directly through the particle sorting device 100 for sorting.

Next, the experimental results will be described with reference to FIGS. 9 to 15.
9 to 15 are diagrams showing specific examples of experimental results. FIG. 9 shows the results of sorting of vegetative cells. FIG. 10 shows the results of sorting spores. In FIGS. 9 and 10, the sorting region of the vegetative cell is region B. In FIGS. 9 and 10, the sorting region of spore is region A. FIG. 9 shows that the density of the region B is high, and FIG. 10 shows that the density of the region A is high.

The vegetative cells were sorted on a polycarbonate membrane having a pore size of 0.2 μm, stained with SYBR Green I, and observed under a microscope. The results are shown in FIG. FIG. 11 is a view taken through a 40-magnification objective lens.
After sorting Vegitative cells, incubate overnight. The culture solution having the number of sorted cells “1” was stained with SYBR Green I, and the results of microscopic observation are shown in FIG. FIG. 12 is a view taken through a 40-magnification objective lens.

After sorting Spore on the membrane, it was stained with SYBR Green I, and the results of microscopic observation are shown in FIG. FIG. 13 is a view taken through a 40-magnification objective lens.
After sorting Spore, incubate overnight. FIG. 14 shows the results of staining the culture medium having the number of sorted cells “1” with SYBR Green I and observing under a microscope. FIG. 14 is a view taken through a 40-magnification objective lens.

The measurement result of the ratio of living cells (result of sorting into a liquid medium) is shown in FIG. As shown in FIG. 15, it can be seen that not all of the cells in the sorting spore solution could be cultured.
-Total bacterial count of spore solution for sorting (counting by microscope): 1.34 x 10 ^ 6 cells / mL
-Number of cells that can grow on agar medium (NBRC702 composition) (colony count): 9.10 x 10 ^ 5 cells / mL
→ Percentage of culturable cells in sorting spore solution: 64.6%

From the above, 64.6% of living cells were originally contained in the spore solution, and 60.4% of the cells were alive as a result of sorting using the spore solution. That is, when the ratio of living cells before and after the sorting operation was compared, it was 93.5%, and it can be seen that most of the spores were not affected by the sorting. In vegetative cells, 100% of the cells were alive as a result of sorting. That is, the ratio of viable cells after sorting is 100%, and it can be seen that vegetative cells are not affected by sorting.

<Modification example>
In the present embodiment, as the laser device 11, the first laser device 11-1 that continuously outputs the laser light in the visible light region, the second laser device 11-2 that continuously outputs the laser light in the deep ultraviolet light region, and the ultraviolet light. Although the three third laser devices 11-3 that continuously output the laser beam in the optical region are shown, the present invention is not limited to these. For example, as the laser device 11, only one laser device 11 (for example, a second laser device 11-2) that continuously outputs a laser beam in a deep ultraviolet light region may be used. As long as the laser device 11 that continuously outputs the laser light in the deep ultraviolet light region is included, the combination of the laser light regions output by the other laser device 11 may be any combination of regions.

In the present embodiment, the well plate has been described as an example as a mechanism for collecting the charged droplets 3, but the mechanism for collecting the droplets 3 may be a tube or a plastic container.
In the present embodiment, the particles have been described as an example of particles that have not been dyed or cannot be dyed. However, the particles used in the particle sorter 100 may be dyed particles.

Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes designs and the like within a range that does not deviate from the gist of the present invention.

The present invention can be applied to particle detection technology.

10 ... Nozzle, 11 ... Laser device, 11-1 ... 1st laser device, 11-2 ... 2nd laser device, 11-3 ... 3rd laser device, 12-1, 12-2, 12-3 ... Focus lens , 13 ... Detection unit, 14 ... Control circuit, 15 ... Display unit, 16 ... Deflection plate, 17 ... Waste liquid tank, 18 ... Well plate, 19 ... Imaging device, 131 ... Objective lens, 132 ... Tube lens, 133 ... Pinhole Plate, 134 ... Prism, 135-1, 135-2, 135-3 ... Collimation lens, 136-1 ... 1st POD, 136-2 ... 2nd POD, 136-3 ... 3rd POD, 137-142, 150-152, 157-159 ... Dycroic mirror, 143-149, 153-156, 160-163 ... Absorption filter

Claims (6)

1. A laser device that continuously outputs laser light in the deep ultraviolet region, and
   A detection unit that detects the fluorescence of the particles obtained by irradiating the particles with the laser light in the deep ultraviolet light region output from the laser device.
   A control circuit that separates the particles based on the detected fluorescence, and
   A particle sorter comprising.
2. The particle sorting device according to claim 1, wherein the deep ultraviolet light region is a region in the deep ultraviolet light region where autofluorescence due to an inorganic mineral is not observed.
3. The particle sorting device according to claim 1 or 2, wherein the deep ultraviolet light region is a region near 230 nm.
4. The particle sorting device according to any one of claims 1 to 3, wherein the particles are discharged by a jet-in-air method.
5. A detection step for detecting the fluorescence of the particles obtained by irradiating the particles with the laser light in the deep ultraviolet light region output from the laser device that continuously outputs the laser light in the deep ultraviolet light region.
   A preparative step of preparating the particles based on the detected fluorescence, and
   Particle sorting method having.
6. A detection step for detecting the fluorescence of the particles obtained by irradiating the particles with the laser light in the deep ultraviolet light region output from the laser device that continuously outputs the laser light in the deep ultraviolet light region.
   A preparative step of preparating the particles based on the detected fluorescence, and
   A computer program that lets a computer run.

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