JPH0824630A - Method for catching and separating fine particles - Google Patents

Abstract

PURPOSE:To enhance the efficiency of the microscopic operation for fine particles such as cell fusion by controlling a deflection means the repeating pulse of a laser beam source so as to individually fix a plurality of particles to a predetermined position to control the laser beam irradiation pattern on a particle present surface. CONSTITUTION:When laser beam is emitted from a semiconductor laser 1 source 11, pulse driving is started in synchronously with the positions of galvano- mirrors 16, 17. At the same time, the galvano-mirrors 16, 17 are also cyclically and repeatedly shaken by the drive current by galvano-drive circuits 14, 15 and an intermittent irradiation pattern is formed in the visual field microscope by laser beam. At this time, the width of the irradiation pattern is set to the same degree as the diameter of desired particles P or less and, when scanning is repeatedly applied to a soln. S, the particles P enter a top site to be caught. By setting the width of a laser pulse to the size of the particles P to be caught and setting a cycle to the interval with the adjacent particle P, the particles P with a desired size are caught one at a time under the visual field of the microscope at a desired interval.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for capturing and separating fine particles in a solution for capturing and separating them.

[0002]

2. Description of the Related Art As means for trapping and moving fine particles in a solution under a microscope field, for example, JP-A-3-110.
An optical trap technique using a laser beam as shown in Japanese Patent Publication No. 510 is known. Examples of application of these techniques include separation of cells floating in a culture solution or cell fusion. This is done by selecting particles using information obtained from microscopic images of floating cells and capturing and moving only the desired particles by an optical trap.

[0003]

However, in order to efficiently perform work such as cell separation, it is desired to capture a plurality of particles at the same time. However, in the conventional method, a plurality of laser beams are used for this purpose. Each must be focused under the field of view of the microscope using a light source, which complicates the device,
There is a problem that the number of particles that can be captured is limited.
Or if you try to move each particle independently,
It is necessary to change the irradiation position for each beam by using a deflecting means, and there is a problem that the optical system for that is complicated.

Further, by detecting scattered light at the time of trapping, the necessity of particles at each site is determined based on the signal, and scanning is performed so that only the trapping sites where the desired particles are present are trapped. , Desired particles can be separated from plural kinds of particles.

An object of the present invention is to provide a method for capturing and separating microparticles that can easily improve the efficiency of micromanipulation of microparticles such as cell fusion with a device having a simple structure.

[0006]

A method for trapping fine particles according to a first aspect of the invention for achieving the above object is a laser light source for intermittently generating a laser beam and a deflecting means to disperse the particles on a surface where particles are present. In a method for trapping microparticles using laser light trapping to generate a specific irradiation pattern,
It is characterized in that the laser light irradiation pattern on the particle existing surface is controlled by controlling the repeating pulse of the deflection means and the laser light source so as to individually fix a plurality of particles at a predetermined position.

Further, the method for separating fine particles according to the second aspect of the invention detects a signal obtained from a plurality of particles that are trapped and switches between trapping and releasing each particle by irradiating laser light using the signals. It is characterized in that a desired kind of particles is separated from a plurality of kinds of particles by carrying out.

[0008]

The fine particle trapping method having the above-described structure controls the irradiation length of the laser pulse and the scanning of the laser beam to slightly change the irradiation position of the laser beam for each scanning, thereby capturing the trapped particles. To move.

Further, in the method of separating fine particles, desired particles are separated by irradiating the particles with a laser beam in accordance with a signal obtained from the particles being trapped to switch between trapping and releasing.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail based on the illustrated embodiments. FIG. 1 is a block diagram of the first embodiment, and 11 is a semiconductor laser light source, which emits laser light having a wavelength of 680 nm, for example. The semiconductor laser light source 11 is controlled by the drive circuit 12 and can emit a laser beam of a pulse up to 1 MHz. In the optical path of the laser light emitted from the semiconductor laser light source 11, a collimator lens 13,
Galvano mirrors 16, 17 driven by the galvano drive circuits 14, 15 respectively, a dichroic mirror 18 having a characteristic of reflecting light having a wavelength of 600 nm or more and transmitting light of other wavelengths, an objective lens 19 of a microscope, a large number of objects to be captured. A solution S containing fine particles P is arranged. This solution S is dripped onto the slide glass 20 and the cover glass 2
Covered by 1. The particles P in the solution S are made of cells or polystyrene having a size of several μm to several 100 μm, and are present in the solution S in a cloudy or floating state.

Below the slide glass 20, 600n
A dichroic mirror 22, a condenser lens 23, and a white light source 2 that transmit light having a wavelength of m or more and reflect light having other wavelengths.
4 are arranged. In addition, the dichroic mirror 22
A lens 25, for example, a photoelectric conversion element 26 composed of a photomultiplier is arranged in the reflection direction, and the output of the photoelectric conversion element 26 is connected to an observation means 27 such as an oscilloscope.

On the other hand, the solution S of the dichroic mirror 18
A lens 30, a barrier filter 31 for transmitting light having a wavelength of 650 nm or less, and a CCD camera 32 are connected in the transmission direction of the luminous flux from the CCD camera 32. The output of the CCD camera 32 is an image processing device 33, a VTR 34, and a television monitor 3.
5 are sequentially connected.

When laser light is emitted from the semiconductor laser light source 11, it is first incident on the galvano mirrors 16 and 17 and is deflected. These laser lights are synchronized with the positions of the galvano mirrors 16 and 17 and Start pulse driving as shown in 2 (a). At the same time, galvanometer mirror 1
6 and 17, when the oscillations are periodically repeated by the driving currents of the galvano drive circuits 14 and 15 as shown in FIGS. 2B and 2C, the shaft 16A including the paper surface and the shaft 1 perpendicular to the paper surface, respectively.
The laser light is swung at a predetermined angle around 7A, and the laser light is, for example, 10 mS as shown in FIG.
Every other intermittent irradiation pattern is formed.

The width g of the irradiation pattern at this time is determined by the laser pulse width e and the angular velocities of the galvano mirrors 16 and 17, and these are set appropriately so that the width g is equal to or smaller than the diameter of the desired particle. And the solution S is intermittently repeated. Then, the particles P moving by the Brownian motion in the solution S enter the trap site and are captured. Since the width g of the irradiation pattern is about the same as or smaller than the diameter of the particle P, the number of particles P captured at each capture site is limited to one, and after several tens of seconds, the particles P are individually separated as shown in FIG. Will be captured in the separated state.

In this state, the lower side is illuminated by the white light source 24, and the image of the particle P is photographed by the CCD camera 32 through the objective lens 19, the dichroic mirror 18, and the barrier filter 31, and is image-processed by the image processing device 33. Then, it is recorded on the VTR 34 at the same time as it is observed by the operator on the television monitor 35. At that time, the barrier filter 31 transmits only the light having a wavelength of 650 nm or less so that the reflected light of the laser light from the particles P does not enter the CCD camera 32.

At this time, the laser pulse width e is set according to the size of the particles P to be captured, and the period f is set appropriately according to the interval between the particles P adjacent to each other, so that the particles P each having a desired size are obtained one by one. , Can be captured under the microscope field at desired intervals. Further, in order to efficiently capture the particles P, the diameter h of the laser light perpendicular to the scanning direction may be changed according to the diameter of the particles P as shown in FIG.

By thus performing the particle trapping, a plurality of particles P can be trapped at desired positions one by one with a device having a simple structure. Also, the capture position can be changed by the laser light irradiation timing or the galvano drive circuit 1.
This can be easily performed by changing the outputs from 4 and 15.

The scattered light of the laser light from the particles P when the laser light traps the fine particles P is reflected by the dichroic mirror 22 and is converted by the photoelectric conversion element 26 via the lens 25 into an electric signal corresponding to the scattered light intensity. Converted,
It is observed by an observation means 27 such as an oscilloscope.

In this case, an electric signal as shown in FIG. 2 (d) is obtained from the photoelectric conversion element 41. It is known that the intensity of the scattered light is mainly determined by the size of the surface reflection of the particle P and the scattering inside the particle P, and the intensity distribution of the scattered light is known to depend on the size of the size of the particle P. Alternatively, when particles P having different sizes are mixedly present in the solution S, it is possible to determine where the particles P are and what kind of particles P by using these pieces of information.

For that purpose, in the state as shown in FIG. 4 in which the particles P are captured at each site, it is compared with preset threshold values V1 and V2 as shown in FIG. 5 (d). As shown in FIG. 5A and FIG. 6, the laser light is driven so that only the sites where the signal intensity from the optical conversion element 26 is judged to be larger than the threshold value V1 and smaller than the threshold value V2 are turned on. Then, after a few seconds, the particles P at the site not irradiated with the laser light move away from the trapping site due to Brownian motion, and as a result, only the desired particles P are trapped as shown in FIG. 7.

At this time, in order to perform more reliable classification,
Noise included in the signal can be reduced by superimposing the output of the optical conversion element 26 for each scanning cycle and averaging the outputs. In addition, by analyzing the obtained waveform, it can be used to judge the classification of the particles P at each site.

By such a method, the particles P can be efficiently separated with a device having a simple structure. In addition, by using the signal obtained by the trap beam acting on the particle P, complicated signal processing such as image processing can be performed,
Separation can be performed by obtaining signals of various optical characteristics of the particle P such as the size of the particle P and scattering on the surface and inside.

Further, the trapped particles P can be moved within the field of view of the microscope by slightly changing the laser light irradiation position for each scanning. The laser light irradiation position is changed by changing the laser light irradiation timing. Alternatively, it can be performed by controlling the galvanometer mirrors 16 and 17 that deflect the laser light.

For example, when performing cell fusion, after capturing and separating cells having desired characteristics as described above, the irradiation pattern of laser light is gradually changed so that two cells are separated as shown in FIG. Put in a proper contact state. Where 360
Cell fusion can be performed by irradiating the contact surface of the cell with a laser beam of nm.

By using such a method, a plurality of particles P can be independently moved one by one with an apparatus having a simple structure, and the particles P can be efficiently operated under a microscope.

In the first embodiment, the scanning was performed during the laser light irradiation, but a galvano drive current as shown in FIG. 9 (b) was applied to make the scanning stepwise, and the laser light was irradiated. Even if the scanning is not performed in the
Can be fixed at desired position one by one. At this time, the pattern of laser light irradiation is as shown in FIG. 10, but the size of the particles P to be captured can be selected by adjusting the diameter i of the laser light.

Further, in the first embodiment, the signal of the optical conversion element 26 is used as the signal for separating the particles P.
It is also possible to take an image with the camera 32, measure the signal such as scattered light intensity at each site by image processing, and use it as a criterion for classification determination.

The signal for separating the particles P may be not only scattered light but also other signals. For example, when classifying floating cells in a culture medium, it is possible to classify and separate cells by fluorescently labeling the cells with an antigen antibody and detecting the fluorescence excited by the trapping beam. is there. Further, when the excitation wavelength of the fluorescent label dye and the wavelength of the trapping laser light are significantly different,
It is also possible to perform similar separation by irradiating the cells with light having an appropriate wavelength using another light source while exciting the cells and exciting and measuring fluorescence.

Although the Brownian motion is used in the above embodiments, it is also possible to accelerate the separation of the particles P by increasing the motion of the particles P with a structure in which the solution S forms a flow.

Further, as a method of capturing the particles P, the particles P can be separated by another method that can fix the particles P individually, instead of scanning with laser light. For example, a plurality of CW laser lights are collected at different positions on the plane of the solution S, and each beam is turned on / off based on a signal from each particle P to separate the particles P. You can also The on / off of the beam may be controlled by a laser drive current, or may be controlled by a liquid crystal shutter capable of independently shielding each beam.

The light source does not have to be a semiconductor laser light source. For example, if an acousto-optic device (AOM) is used, a CW laser is used to obtain laser light of repetitive pulses, and particles P are similarly captured and separated. It is possible.

[0032]

As described above, in the method for capturing fine particles according to the first aspect of the present invention, a plurality of positions for irradiating laser light are
By controlling the irradiation length of the laser pulse and the scanning of the laser light so that the size is the same as or smaller than the size of the trapped microparticles, the microparticles can be moved to the desired position.
It becomes possible to capture them one by one.

Further, in the method for separating fine particles according to the second aspect of the present invention, the particles are irradiated with a laser beam according to a signal obtained from the particles that have been trapped, and the trapping and releasing of the particles are switched to separate the desired particles. be able to.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a first embodiment.

FIG. 2 is a waveform diagram of signals from laser driving, scanning system driving, and each particle.

FIG. 3 is an explanatory diagram of a scanning state under a microscope field of view and an irradiation position of laser light.

FIG. 4 is an explanatory view of a state in which particles are trapped at each trapping site.

FIG. 5 is a waveform diagram of signals from laser driving, scanning system driving, and each particle.

FIG. 6 is an explanatory diagram of a laser light irradiation position.

FIG. 7 is an explanatory diagram of a state in which only desired particles are captured.

FIG. 8 is an explanatory diagram of a state where particles are moved.

FIG. 9 is a waveform diagram of laser driving and scanning system driving in the second embodiment.

FIG. 10 is an explanatory diagram of a laser light irradiation position.

[Explanation of symbols]

 11 semiconductor laser light source 12 laser drive circuit 16, 17 galvano mirror 18 dichroic mirror 19 objective lens 26 photoelectric conversion element 32 CCD 33 image processing device

Claims (5)

[Claims] 1. A method for trapping fine particles using laser light trapping, wherein a laser light source for intermittently generating laser light and a deflecting means generate a discrete irradiation pattern on a surface on which particles are present. A method for capturing fine particles, characterized in that a laser light irradiation pattern on a particle existing surface is controlled by controlling a repeating pulse of the deflecting means and the laser light source so as to individually fix the particles at a predetermined position. 2. The method for capturing microparticles according to claim 1, wherein the size of the laser beam irradiation pattern is substantially equal to or smaller than the size of particles captured at each particle capturing site. 3. The particles are moved by controlling the timing of laser light irradiation or the deflection means to change the position of laser light irradiation on the surface where the particles are present for each scanning. The method for capturing microparticles according to [1]. 4. A signal obtained from a plurality of particles being trapped is detected, and switching between trapping and releasing of each particle is performed by irradiating a laser beam using the signal to obtain a desired signal from a plurality of types of particles. A method for separating minute particles, characterized in that particles of different types are separated. 5. The method for separating microparticles according to claim 4, wherein the signal obtained from the particles is a signal obtained by a laser beam that traps the particles acting on the particles.