JPH01313758A - Cell sorting device - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an improvement in a cell sorter (cell sorter) that destroys and kills unnecessary cells using a laser.

(b) Prior art In recent years, in order to improve the accuracy of cell sorting, a cell sorting device that uses a laser to destroy and kill unnecessary cells has been proposed. The configuration and operation of this cell sorting device will be explained with reference to FIGS. 6 and 7.

C is a cell flowing in a fine water stream, and this cell C is irradiated with the detection laser beam 11 from the detection laser 33 at point A, which is focused by the lens system 34. Scattered light or fluorescence (hereinafter referred to as signal light) 12 is induced from the cells C, and this signal light 12 is focused by a condenser lens system 36, received by a photodetector 38, and converted into an electric pulse signal d. [See Figure 7(a)].

The pulse signal d is input to the discrimination circuit 39, and this discrimination circuit 39 determines whether the cell is necessary or not, and if it is not necessary,
A cell killing command pulse e is output to the delay circuit 41. The delay circuit 41 outputs the pulse h to the biocidal laser 40 with a delay of time t, and the time t is set to the time during which the cell C flows from point A to point B where the pulsed biocidal laser beam P3 is irradiated. be done.

When the pulse h is outputted from the delay circuit 41, the biocidal pulse laser beam 13 is irradiated to point B through the optical system 43 in synchronization with this, and the cell C is destroyed and killed. When cells C are needed, the discrimination circuit 39 does not output the pulse e and the biocidal pulsed laser beam 13 is not irradiated, so the cells C simply flow through the water stream.

(c) Problems to be Solved by the Invention In the above-mentioned conventional cell sorting device, when the speed of the water flow fluctuates, when cells C that have passed through point A pass a preset delay time t, , there is a possibility that it does not exist at point B. Further, in the discrimination circuit 39, the criteria for determining whether or not cells are necessary are complex and take time to make the determination, making it difficult to set an accurate delay time t. For this reason, there was a problem in that unnecessary cells could not be reliably destroyed or killed.

In particular, the irradiation time of the pulsed laser beam 13 is a delay time t
The above-mentioned problem becomes even more noticeable when the pulse laser beam 130 is extremely short compared to the above, or when the intensity of the pulsed laser beam 130 is weak and narrowly focused for concentrated irradiation.

This invention was made in view of the above, and aims to provide a cell sorting device that can reliably destroy and kill unnecessary cells. (d) Means for Solving the Problems The structure of the cell sorting device of the present invention will be explained using FIG. 1, which corresponds to one embodiment. Detection laser light hits cells that reach detection point A in liquid flow 2! , and a signal light 12 from the cells irradiated with the detection laser light 11.
The first photoreceiver 8 and 1. A determining means 9 determines whether or not the cells are necessary based on the output signal of the first light receiver 8, and based on the determination result of the determining means 9, In a cell sorting device comprising, at a sorting point B, a biocidal laser 10 that irradiates a biocidal laser beam 13 to cells that have reached the sorting point B, a timing determining laser 11 that irradiates the timing determining laser beam 14 to the sorting point B. and a second light receiver 17 that receives the second signal light l emitted when the cells that have reached the sorting point B are irradiated with the timing determining laser light 14, and the output of this second light receiver 17. The present invention is characterized by comprising a control means 19 for irradiating a biocidal laser beam 13 to cells that have reached the sorting point based on a signal.

(E) Function The cell sorting device of the present invention detects the signal light L generated by the irradiation of the timing determining laser beam 14, and when it knows that the cell has reached the sorting point B, the cell sorting device of the present invention applies the biocidal laser beam to the cell. 13, there is no need to set a delay time, and cells can be reliably destroyed and killed even if fluctuations occur in the flow.

(f) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 5.

FIG. 1 is a diagram illustrating the configuration of a cell sorting device 1 according to an embodiment of the present invention. 2 is a thin water stream formed by means such as a flow cell.

On the central axis of this water flow 2, cells flow in a line due to a so-called hydrodynamic focusing effect.

A detection laser beam from the detection laser 3 is directed to a point in the water flow 2! , is focused and irradiated by the lens system 4. As the detection laser 3, a continuous wave type laser such as a He-Ne laser, an Ar laser, a Kr laser, a He-Cd laser, etc. is applied.

Signal light (scattered light or fluorescence) generated from cell C at point A1
2 is focused by a condenser lens system 6, transmitted through a pinhole 7, and received by a first photodetector 8. The pinhole 7 is for removing scattered light generated by the wall surface of the flow cell, and the reference numeral 5 is a beam stopper for preventing the detection laser beam l from directly entering the photodetector 8. be.

On the other hand, 10 is a pulse laser for cell killing, and Nd:
A solid laser such as YAG and a laser that generates harmonics thereof are desirable. In particular, since ultraviolet light with a wavelength shorter than 300 nm is absorbed without staining cells, lasers that emit such ultraviolet light are suitable for cell killing. Pulsed laser beam ffi from this pulsed laser 10,
(indicated by a broken line) is condensed and irradiated by the optical system 13 to point B downstream from point A.

11 is a pulsed laser beam 2. This is a timing determination laser for determining the timing of l. Although a continuous wave laser is used as the laser 11 for timing determination, it is not necessary to provide the detection laser 3 and the timing laser 11 separately in this way, and the laser beam l of the detection laser 3 is It may be used by dividing it with a beam splitter or the like.

The laser beam 14 (indicated by a solid line) from the laser 11 for timing determination is made to have the same optical axis as the pulsed laser beam 23 by the beam splitter 12, and is also focused on point B.

Laser beam 1 for timing determination is applied to the cell at point B.
The second signal light 2.4 is induced by being irradiated with the second signal light 2.4.
is condensed by the condensing lens system 15 and received by the second photodetector 17. Note that the pinhole 16 is for preventing scattered light from the flow cell, etc., and the pulse laser beam 11 and the timing determination laser beam p4 are directly connected to the photodetector 17.
prevent it from entering.

20 is a container for receiving the effluent that has been subjected to laser irradiation and sorting processing.

The pulse signal from the first photodetector 8 is input to a discrimination circuit 9, and the killing command pulse output from this discrimination circuit 9 is input to a pulse peak detection circuit 18. This pulse peak detection circuit 18 detects the peak of the pulse signal from the second photodetector 17 and outputs the detected pulse signal to the laser control circuit 19. The laser control circuit 19 is
Controls the oscillation of the pulse laser 10.

Next, the operation of the example cell sorting device 1 will be explained with reference to FIG. 2 as well.

Cell C flows down in water stream 2 and when it reaches point A, cell C receives a laser beam! , signal light 12 is induced.

This signal light 12 is transmitted to the first photodetector 8 as shown in FIG. 2(a).
It is converted into a pulse signal d shown in FIG. The discrimination circuit 9 discriminates whether or not the cells are necessary based on this pulse signal d, and if the cells are not necessary, the biocidal command pulse e is sent to the pulse peak detection circuit 1.
This pulse peak detection circuit 18 is driven.

On the other hand, when the cell C reaches point B from point A and crosses the laser beam 14 for timing determination, a second signal light I is induced and received by the second photodetector 17, as shown in FIG. C)
It is converted into a pulse signal r shown in FIG. When the killing command pulse C is input to the pulse peak detection circuit 18, the pulse peak detection circuit 18 detects the signal light 2. When the pulse signal f, which is proportional to the intensity of In addition, Fig. 2 (C)
The medium broken line indicates that the cells were destroyed and killed.

The pulsed laser beam 13, like the laser beam 14 for timing determination, is directed to irradiate the cells at point B, so when the pulse signal f reaches the maximum, that is, the center of the unnecessary cell is at point B. When it reaches, pulse laser beam! , is irradiated to ensure that unnecessary cells are destroyed and killed.

Furthermore, even if it takes time for the discrimination circuit 9 to determine whether cells are necessary or not, if the determination can be made during the period from point A to point B and the peak detection circuit 18 can be driven, unnecessary cells can be eliminated. The pulsed laser beam 13 can be irradiated to destroy and kill the creatures.

If cells are required, the discrimination circuit 9 generates a biocidal command pulse e.
is not output, and the necessary cells simply flow away from point B. capacity
The effluent received in the J''fr20 contains necessary cells and destroyed and killed cells, and the necessary cells can be separated and collected by a simple operation such as a centrifugal bone 1'ilt.

In the cell sorting device 1 of this embodiment, a pulse l/- laser is used as a cell killing laser, but a continuous wave laser can also be used. In this case, when the required cells reach point B, f1, the biocidal laser may be stopped [1:], or an acousto-optic element may be used to shield the laser beam; It is desirable to set the beam irradiation point slightly upstream of the laser beam irradiation point.

FIG. 3 is a block diagram showing a modification of the signal processing system.

In this case, the discrimination circuit 9 and the pulse peak detection circuit 18
A delay circuit 21 is provided between the two.

Based on the pulse signal d from the first photodetector 8 (see FIG. 4 CI), the discrimination circuit 9 issues a killing command pulse e [
(See Figure 4, etc.)] When the pulse h is output to the delay circuit 21, the delay circuit 21 outputs a pulse h that is delayed so as to sufficiently include the time when the unnecessary cell arrives at the position of point B [See Figure 4 (C
)reference〕. The width of this pulse h is 1. is set so that the pulse signal f [see FIG. 4(d)] induced by the laser beam 1.14 for timing determination is sufficiently included even if the flow velocity of the cell flow varies.

When a cell determined to be unnecessary for the discrimination circuit reaches point B, the delayed pulse h drives the pulse peak detection circuit 18. The pulse peak detection circuit 18 outputs a trigger pulse g when the pulse r reaches its peak, and the cells are irradiated with a pulsed laser beam 23 for killing cells, thereby destroying and killing the cells.

In this modification, even if the concentration of cells in the water flow is large and the distance between cells is smaller than the distance between points A and B, the delay time is originally 1. It has the advantage of being able to reliably destroy and kill unnecessary cells by appropriately setting.

FIG. 5 shows a modification of the cell sorting device 1 in which the optical system is simplified. Laser beam 1 for cell detection
1 is focused by the lens system 23 at a point A on the central axis of the flow channel 22a of the flow cell 22. The optical axes of the life-killing pulsed laser beam 13 (indicated by the broken line) and the timing determination laser beam 12 (indicated by the - dotted chain line) overlap, and both are connected to the flow channel 2 by the lens system 23.
The light is focused on point B downstream of point A on the center axis of 2a.

Signal light induced in the cell at point A/! 2 (indicated by a solid line) is condensed by the condensing lens system 24 and filtered by the filter 25.
The light passes through the pinhole 26b and is received by the first photodetector 8. On the other hand, at point B, the signal light e (indicated by the - dotted chain line) induced in the cells is also focused by the condenser lens system 24 and transmitted through the filter 25 and pinhole 26a to the second
The light is received by the photodetector 17. 27 is L nose beam 2. , l3.14 is a beam stopper that prevents the beam from directly entering the photodetector 8.17. Also, filter 2
5 reflects or absorbs the pulsed laser beam scattered by the cells, and the scattered light of the pulsed laser beam is transmitted to the photodetector 8.
．． This is provided for the purpose of preventing the light from entering 17.

(G) As described in detail of the invention, the cell sorting device of the present invention includes a timing determining laser that irradiates a timing determining laser beam to a sorting point, and a timing determining laser that irradiates a timing determining laser beam to a sorting point. A second photodetector receives a second signal light emitted upon irradiation with the light, and based on the output signal of the second photodetector, the cells that have reached the sorting point are irradiated with a biocidal laser beam. Since the device is equipped with a control means for controlling the biocidal laser so that the biocidal laser is It has the advantage of destroying and killing unnecessary cells.

Also, when focusing a small laser beam for killing insects,
Since this focused laser beam can reliably irradiate unnecessary cells, energy can be concentrated on unnecessary cells and cell killing efficiency can be increased. This also has the advantage of making it possible to downsize and lower the cost of the device for differentiating cells jπ.

[Brief explanation of the drawing]

FIG. 1 is a diagram illustrating the configuration of a cell sorting device according to an embodiment of the present invention, FIG. 2 is a time chart explaining the operation of the cell sorting device, and FIG. 3 is a diagram illustrating the operation of the cell sorting device. FIG. 4 is a block diagram showing a modified example of the signal processing system, and FIG. 5 is a block diagram illustrating the operation of the signal processing system.
A sixth diagram illustrating a modification of the optical system of the cell sorting device.
The figure is a diagram explaining the configuration of a conventional cell sorting device, and FIG. 7 is a time chart explaining the operation of the conventional 1llll cell sorting device. 2: Water flow, 3: Detection laser, 8: First photodetector, 9: Discrimination circuit, 10: Pulse laser, 11: Timing determination laser, 17: Second photodetector, 18: Pulse peak detection Circuit, 19: Laser control circuit. Patent Applicant Tateishi Electric Co., Ltd. Agent Patent Attorney Shigeru Nakamura Figure 2 d Figure 3 Figure 4

Claims (1)

[Claims] (1) A liquid flow in which cells flow in a line, a detection laser that irradiates detection laser light to cells that reach a detection point in this liquid flow, and cells that are irradiated with this detection laser light. a first light receiver that receives a signal light; a determining means for determining whether or not the cells are necessary based on the output signal of the first light receiver; A cell sorting device comprising: a biocidal laser that irradiates a biocidal laser beam to cells that have reached a sorting point downstream from a detection point; a timing determining laser that irradiates a timing determining laser beam to the sorting point; , a second photodetector that receives a second signal light emitted when the cells that have reached the sorting point are irradiated with the timing determining laser light; and based on the output signal of the second photodetector, the A cell sorting device comprising: control means for controlling the biocidal laser so that the biocidal laser light is irradiated to cells that have reached a sorting point.