JPH0427866A - Cell sorting apparatus - Google Patents

Abstract

PURPOSE:To certainly kill an unnecessary cell by providing an irradiation interval detection means detecting the irradiation interval of a killing continuous oscillation Q switch pulse laser and a Q control means controlling Q of the laser resonator of the Q switch pulse laser on the basis of the detected irradiation interval. CONSTITUTION:A fine liquid stream 2 is allowed to flow in a row by a means such as a flow cell and discriminating laser beam l1 is condensed and applied to the detection point A in the liquid stream 2 from a discriminating laser and the forward scattering beam l4 generated by a cell C at the point A is condensed by a lens system 8 to be detected by a photodetector 12. The orthogonal signal beam l7 such as scattering beam or fluorescence emitted to the laser beam l1 generated from the cell C at the point A in an orthogonal direction is condensed by a lens system 9 and sorted by a filter 10 to be detected by an orthogonal beam detector 13. The laser beam l3 from a cell killing pulse laser 4 is condensed and applied to the sorting point B on the downstream side of the point A and a part of the beam is split by a beam splitter 6 to be detected by a pulse laser beam detector 15 and an unnecessary cell is killed by a Q control means while a necessary cell is left.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a cell sorting device (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, cell sorting devices have been proposed that use a laser to destroy and kill unnecessary cells. The configuration and operation of this cell sorting device will be explained with reference to FIGS. 5 and 6.

C is a cell flowing in a thin liquid stream, and this cell C is illuminated by the discrimination laser beam from the discrimination laser 33 at point A! 1 is focused by a lens system 34 and irradiated. Scattered light or fluorescence (hereinafter referred to as signal light) from cell C1! is induced, and this signal light 12 is focused by a condenser lens system 36, received by a photodetector 3, and converted into an electric pulse signal d (see FIG. 5).

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, but at time t, when the cell C is from point A,
Pulsed laser light for life-killing! 3 is set to the time it takes to reach point B where it is irradiated.

When the pulse h is output from the delay circuit 41, a pulsed laser beam for life-killing is produced in synchronization with this! 3 through the optical system 43
The point is irradiated, and cells C are destroyed and killed. When cell C is needed, pulse e is not output from the discrimination circuit 39, and the pulsed laser beam for killing biocides! , are not irradiated, so the cells C flow as they are in the liquid flow.

(C) Problems to be Solved by the Invention In the conventional cell sorting apparatus described above, a continuous wave (CW) Q-switched pulsed laser is often used as the biocidal laser 40. This CW, Q-switched pulse laser is
An optically excited solid-state laser such as a CW or YAG laser is converted into pulsed light and emitted by a method called Q-sinch using an acousto-optic device.

As a general characteristic of this type of laser, the intensity of the pulsed laser light varies depending on the pulse interval h, that is, the repetition frequency of pulsed laser irradiation. This fluctuation causes the intensity of the pulsed laser beam to fluctuate as the repetition frequency increases. In other words, the shorter the time interval between previous irradiations, the lower the intensity of the pulsed laser light.

This phenomenon will be explained in detail using FIG. A fixed value is used for the Q of the pulsed laser resonator (Q
,=Qz). Subscript 1 indicates the laser pulses irradiated at sufficient time intervals, and subscript 2 indicates the laser pulses I.
Assume that laser pulses (3) are emitted at shorter intervals.

nil, n LI% n r+ are the initials of the number of excited state active substances (PI) of the laser pulse ■, respectively;
It shows the threshold and final. The difference between nil and nfl corresponds to the intensity of the laser beam, which is 1.

n1□, nfl, nfZ are laser pulse 2 as before.
P [ is shown. However, the time interval.

Since PI is short, the PI that has fallen to n, fl returns only to ni2, which is lower than the original nil. Moreover, when the laser pulse ■ is irradiated in this state, the PI drops to the previous n.
It only drops to nrz, which is a higher value than fl. Therefore, niz nfZ is nil nfl
The intensity L2 of the laser pulse H becomes smaller than the intensity L2 of the laser pulse (2).

In this way, if the time interval between laser pulse irradiations becomes short and the laser light intensity decreases, unnecessary cells cannot be completely killed. On the other hand, if the irradiation interval becomes longer, excessively intense laser pulses may be irradiated, damaging the optical components that make up the device, or even necessary cells may be irradiated with scattered laser light and damaged. There was a problem.

This invention was made in view of the above, and aims to provide a cell sorting device that can reliably kill unnecessary cells and does not damage necessary cells or optical components.

(d) Means for solving the problems In order to solve the above problems, the cell sorting device of the present invention includes:
It has the configuration described in items 1 to 2 below.

: A liquid stream in which cells flow in a line, 11: A discrimination laser that irradiates a discrimination laser beam to cells that have reached a detection point in this liquid flow, and 111: Irradiates this discrimination laser beam. a photodetector that receives a signal light from a cell that has been detected; 1): a determining means that determines whether or not the cell is necessary based on the output signal of the photodetector; and V: this determining means outputs a determination signal. a delay means for outputting a trigger signal when a predetermined delay time has elapsed; i: upon receiving this trigger signal, a biocidal pulse is applied to cells that have reached a sorting point downstream of the detection point in the liquid flow; In the device comprising a biocidal CW and a Q-switch pulse laser for irradiating laser light, VTi: irradiation interval detection means for detecting the irradiation interval of the biocidal CW and Q-switch pulse laser, and vii: detection of this irradiation interval. Q control means for controlling the Q of the laser resonator of the CW, Q-sinch pulse laser based on the irradiation interval detected by the means.

(E) Function The function of the cell analysis device of the present invention will be explained using FIG. The case shown by the broken line in FIG. 5 is the case of this invention. If CW, Q-switched pulsed lasers are irradiated with sufficient time intervals, keep the Q low.
(Q,'). If Q is low, the drop in PI is controlled (n'=+n'r+(<ni+-nrI)),
The intensity of the laser beam does not become excessively large (L+'<
L+). Furthermore, since n'r+>nfl, PI recovery is also faster.

When laser pulses ■ are irradiated at even shorter time intervals, Q is increased (Qz'>(Q+')),
The drop of pr is increased (n'tz n'rz (kn, □2nrz)), and the laser light intensity L2 is maintained at the level necessary for killing the creatures.

Therefore, if a predetermined relationship is established between the irradiation interval and Q, it is possible to always irradiate a laser pulse with uniform intensity, regardless of the length of the irradiation interval.

(F) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 4.

FIG. 2 is a perspective view illustrating the optical system of the cell sorting device of this example. 2 is a thin liquid stream formed by means such as a flow cell. On the central axis of this liquid flow 2, cells flow in a line due to a so-called hydrodynamic focusing effect.

One point (detection point) A in the liquid flow 2 is focused and irradiated with a discrimination laser beam 21 from a discrimination laser (not shown). Continuous wave lasers such as a HeNe laser, an Ar laser, a Kr laser, a He-ca laser, and a semiconductor laser are used as the discrimination laser.

Forward scattered light generated from cell C at point A! , is focused by the front condensing lens system 8 and received by the photodetector 12 for forward scattering. 7 is a laser beam for discrimination! 1 is a beam stopper for preventing the light from directly entering the photodetector 12.

Laser beam generated from cell C at point A2. Orthogonal signal light j27, such as scattered light or fluorescence emitted in a direction perpendicular to , is collected by a right-angle condenser lens system 9, sorted by a filter 10, and then received by a right-angle light detector 13.

On the other hand, 4 is a pulse laser for cell killing, CW, Q
A switch, a solid-state laser such as Nd: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. The pulsed laser beam 13 from this pulsed laser 4 is focused and irradiated to point B (selecting point) downstream from point A.

This pulsed laser light! , is split by the beam splitter 6 and received by the pulsed laser beam photodetector 15.

14 is pulsed laser light! This is a laser beam for monitoring the position of cells and for adjusting the timing of firing. A continuous wave laser is used as the position monitoring laser (not shown), but it is not necessary to provide separate lasers for discrimination and position monitoring, and the laser beam of the discrimination laser p1 may be divided and used using a beam splinter or the like.

This laser beam p4 for position monitoring is a pulsed laser beam! , and the light is focused on point B. Monitoring signal light induced by irradiating cells with position monitoring laser light r4 at point B! ,
is received by the monitoring photodetector 14. The beam stopper 7 is a laser beam for monitoring this position! 4 from directly entering the monitoring photodetector 14.

Reference numeral 11 denotes a container for receiving the effluent that has been irradiated with the biocidal laser beam 23 and has been subjected to the sorting process.

Next, the circuit configuration of the cell sorting device according to the embodiment will be explained with reference mainly to FIG. Note that the arrangement of the photodetectors in FIG. 1 is for convenience of explanation and does not represent the actual arrangement.

The pulse signal output S of the photodetector 12 for forward scattered light is transmitted through an oscilloscope 16 for displaying a signal waveform, a peak detection circuit 17 for detecting that a cell has passed through the center of the discrimination laser beam II, and a cell essential point. - Input to the discriminating circuit 21 for discriminating unnecessary.

In addition to the pulse signal S of the forward scattered light detector 12, the discrimination circuit 21 also receives a signal from the orthogonal light photodetector 13,
It is determined whether the cells passing through point A are necessary or unnecessary, and when the cells are unnecessary, a determination pulse D is output to the ink pulse counter 22 and the AND circuit 19.

In the interval count 22, the interval from the rise of the previous cell discrimination pulse to the rise of the current discrimination pulse is counted based on the clock pulse of the built-in oscillator. The count number of the ink pal counter 22 is output to the lookup table 23.

The lookup table 23 corresponds to the rising interval of the input discrimination pulse and determines the Q of the resonator during laser pulse irradiation of the biocidal pulse laser 4. 24, and this DAC 24 holds the amplitude level voltage.

The contents of this lookup table 23 are shown in FIG. FIG. 4 shows the case where the clock frequency of the interval counter 22 is IMHz, the horizontal axis shows the count number n of the interval counter 220, and the subaxis shows the modulation level (%). In this embodiment, a ROM is used as the lookup table 23, but it may also be configured with a RAM and the contents of the table may be read into the RAM when the apparatus is started.

On the other hand, the output of the peak detection circuit 17 is
Enter. The delay time set for one day in the Tilley circuit is adjusted so that it is approximately equal to the time required for the cell to arrive from point A to point B.

The output pulse Pd of the delay circuit and the output of the discrimination circuit 21 are input to an AND circuit 19, and the AND circuit outputs a pulse to the gate pulse generation circuit 20 only when both inputs are positive. This gate pulse generation circuit 20 generates a gate pulse Ct that determines the time to raise the Q of the laser resonator in order to irradiate the laser pulse.

This gate pulse Gt is sent to the DAC 24 by the synthesis circuit 25.
It is combined with the amplitude level voltage A of , and is used as a modulation signal MD. This modulation signal MD is transmitted to the modulator 26 and the RF oscillator 27.
It modulates a higher frequency (usually around 50M old). This modulated RF is input to an acousto-optic element 28 for controlling the Q of laser resonance of the pulsed laser 4.

The oscilloscope 16 synchronously displays the output S of the forward scattered light detector 12, the output M of the position monitoring photodetector 14, and the output of the pulsed laser photodetector 14. While looking at the screen 16a of the oscilloscope 16, the operator adjusts the delay so that the output waveform of the pulsed laser photodetector 15 matches the output waveform M of the cell position monitoring photodetector 14 on the time axis. Adjust the circuit 18 and use the laser beam to kill unnecessary cells! , to ensure that it is irradiated.

Next, the operation of the cell sorting device of this embodiment will be explained with reference mainly to the time chart shown in FIG.

When a cell reaches point A, this cell emits signal light p, ,
(see FIG. 2), the forward scattered light detector 12 and the right angle light detector 513 output signals S1, respectively.
R+ (not disclosed) is output. Signal S, ,
R, is input to the discrimination circuit 21, and arithmetic processing for discrimination is performed. Note that this arithmetic processing is not the main part of the present invention, so the details will be omitted.

The signal S is also input to the peak detection circuit 17, and the signal S
, a peak signal P is detected.

is output. The peak signal P is input to the delay circuit 18, and after a set delay time has elapsed, a delay pulse Pd is output.

On the other hand, the discrimination pulse D1 rises from the discrimination circuit 21 after the time required for the calculation. When the determination pulse D1 rises, the count number n of the interval counter 22 is output to the look amplifier table 23, and the ink pulse counter 22 is reset to start counting anew from 1. Lookup Chiful 23 is the 4th
From the relationship shown in the figure, the modulation level corresponding to nl is determined by the DAC2.
Output to 4. The DAC 24 outputs the amplitude level voltage A to the synthesis circuit 25.

Since the AND circuit 19 receives both the delay pulse Pd+ and the discrimination pulse D, it outputs a pulse that is the logical product of the delay pulse Pd+ and the discrimination pulse D, which is converted into a gate pulse Gt+ by the gate pulse generation circuit 20.

This gate pulse Gt+ is input to the synthesis circuit 25,
It is synthesized with the amplitude level A, and outputs a modulated RF wave signal MD.

The output of the RF oscillator 27 is sent to the modulator 26 as a modulation signal MD,
is modulated and input to the acousto-optic element 28, and the Q of the laser resonator is set to Q. After the end of the previous laser emission,
In the charged active substance, the Q of the laser resonator changes from the high loss QL state to the low loss Ql state, and the biocidal laser beam 13 is emitted. The laser output reaches its maximum when the number PI of active substances in an excited state reaches a threshold value n't+ (see FIG. 5).

Life-killing laser light! 3 is detected by the photodetector 15 for biocidal pulsed laser beam, and this photodetector 15 for pulsed laser beam
The pulsed laser light signal L1 can be observed with an oscilloscope 16. Note that the discrimination pulse D1 falls at the same time as the delay pulse Pd.

In FIG. 3, subscripts Q and 2 indicate cells before and after the cell that generated the signal S1 (both are unnecessary). The irradiation interval of the signals S0 and S2 is the signal S, , S
, is smaller than the irradiation interval of .

Therefore, n2<n, so from the lookup table in FIG. 4, the modulation level for signal S2 is
It will be lower than in case 1. Therefore, the amplitude level is set high < (AI) for the signal S, and low (A2) for the signal S2. Therefore, in the case of the signal S1, Ql is set low, and conversely, in the case of the signal S2, Q2 is set high, and the life-killing pulsed laser beam of almost uniform intensity is irradiated regardless of the irradiation interval.

In FIG. 3, signal S3 is for the cells of interest.

In this case, since no discrimination pulse is output, the interval counter 22 continues to count. Also AND
The circuit 19 does not output a pulse, and the gate pulse generation circuit 2
0 also does not output a gate pulse.

Signal M is the output signal of the position monitoring photodetector 14 in FIG. The caustic line on the right half of ,M, ,M, is a pulsed laser beam that kills cells! 3 indicates that it was destroyed.

Therefore, using the oscilloscope 16, it can be confirmed whether or not unnecessary cells have been reliably destroyed.

In this embodiment, the output signal of the forward scattered light photodetector 12 is input to the peak detection circuit 17, but a configuration in which the output signal of the orthogonal light detector 13 is input to the peak detection circuit 7 is also possible. Good too.

(g) As described in detail of the invention, the cell sorting device of the present invention includes an irradiation interval detection means for detecting the irradiation interval of the biocidal CW and Q-switched pulsed lasers, and an irradiation interval detection means for detecting the irradiation interval of the biocidal CW and Q-switched pulse lasers. The present invention is characterized by comprising a Q control means for controlling the Q of the laser resonator of the biocidal CW and Q-sinch pulse laser based on time. Therefore, biocidal CW.

The pulse laser light intensity of the Q-sinch pulse laser is uniform regardless of the irradiation interval, which has the advantage of being able to reliably kill unnecessary cells and not damaging necessary cells or optical components.

[Brief explanation of drawings]

FIG. 1 is a block diagram explaining the circuit configuration of a cell sorting device according to an embodiment of the present invention, FIG. 2 is a diagram explaining the optical system of the cell sorting device, and FIG. Figure 4 is a time chart explaining the operation of the device, and Figure 5 is a diagram explaining the contents of the lookup check of the cell sorting device.
The figure is a time chart explaining the operation of the present invention and the problems of the conventional cell sorting device, and FIG. 6 is a block diagram explaining the configuration of the conventional cell sorting device. 2: Liquid flow, 4: Biocidal pulse laser, : Photodetector for forward scattered light, : Photodetector for right-angle light, : Delay circuit, 21: Discrimination circuit, : Interhull counter, : Lookup table, Bisynthesis Circuit, 26: Modulator, : Acousto-optic element.

Claims (1)

[Claims] (1) A liquid flow in which cells flow in a line, a discrimination laser that irradiates discrimination laser light to cells that reach a detection point in this liquid flow, and cells that are irradiated with this discrimination laser light. a photodetector that receives a signal light; a determining means that determines whether or not the cells are necessary based on the output signal of the photodetector; and a predetermined delay time elapses after the determining means outputs the determination signal. and a biocidal CW which receives the trigger signal and irradiates biocidal pulsed laser light to cells that have reached a sorting point downstream of the detection point in the liquid flow. In a cell sorting device comprising a Q-switched pulsed laser, the biocidal CW. The CW. A cell sorting device comprising: Q control means for controlling the Q of a laser resonator of a Q-switched pulsed laser.