JPH0728714B2 - Multi-beam type small object processing device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention mainly relates to a micro-object processing apparatus for capturing, sorting, and isolating cells / intracellular organelles, and more particularly to cells / intracellular organelles. The present invention relates to a multi-beam type minute object operation sorting / picking-up apparatus applicable to minute objects such as dielectric particles that can be captured by a laser beam.

[0002]

2. Description of the Related Art Conventionally, when capturing, sorting, or isolating cells or subcellular organelles, while observing the cells under a microscope, a micropipette is moved up, down, left, or right by an actuator. Track the cells you need,
I was capturing. However, tracking of cells and the like, particularly tracking of motile cells requires a high level of technology and there is a problem that they cannot be easily captured.

[0003]

SUMMARY OF THE INVENTION The technical problem of the present invention is to obtain a multi-beam type micro object processing apparatus capable of capturing, sorting and collecting micro objects such as cells and subcellular organelles by a simple operation. Especially.

[0004]

A first micro object processing apparatus of the present invention for solving the above problems is a pair of trapping laser beam projecting means for projecting opposed laser beams for capturing a micro object. And a take-out laser beam projecting means for projecting a laser beam for ejecting a minute object captured by these laser beams from a direction intersecting with the opposing laser beam, and a taking-out laser beam for the minute object. And a control device for controlling the playing of the. Further, the second minute object processing apparatus of the present invention controls the laser beam projecting means for a large number of guideways for projecting the laser beam along the guideways of the minute objects intersecting each other, and controlling the respective laser beam intensities. However, a control device for moving the minute object along the desired guideway side laser beam at the intersection of the guideways is provided.

[0005]

In the first minute object processing apparatus of the present invention,
When the minute objects are sequentially drawn between the facing laser beams and captured, both beams are projected facing each other and are thus held in line on the beam. Then, these minute objects can be ejected by projecting a laser beam for extraction, and are separated and collected by the extraction device arranged ahead of them.

Further, in the second minute object processing apparatus of the present invention, the minute object is moved by being placed on the laser beams along a number of guideways intersecting with each other, and at that time, the intensity of each laser beam is controlled by the control apparatus. The guideway on which the minute object rides is switched by controlling the. As a result, it is possible to move one by one or an arbitrary number of minute objects to the exit of an arbitrary guideway and sort and collect them.

[0007]

Embodiments of the present invention will be described in detail below with reference to the drawings. FIG. 1 shows a first embodiment of a multi-beam type micro object processing apparatus according to the present invention. As the first embodiment, a counter beam type cell manipulation sorting and collection using two counter type laser beams is shown. The structure of an apparatus is shown.

As shown in FIG. 1, the opposed beam type cell manipulation sorting and collecting apparatus according to the first embodiment has opposed laser beams B11 and B12 for capturing cells and subcellular organelles as micro objects 10. Laser beam projection means (not shown) for projecting a laser beam, and a laser beam B13 for ejecting a minute object captured by these laser beams is projected from a direction intersecting the laser beams B11, B12. It is provided with a laser beam projecting means (not shown) for taking out, and further provided with a final taking-out device 18 such as a micropipette.

The above-mentioned opposed laser beams B11 and B12
As the projection means, laser devices are arranged to face each other, and these laser devices form a capture device that captures and holds the minute object 10 in a floating state on the stage 17 of the microscope. The micro object 10 such as a cell captured by this capture device is held in an intermediate position in a non-contact manner based on the principle described later, and can be observed under a microscope as it is. can do.

The laser beam B13 for taking out is projected so as to be orthogonal to the laser beams B11, B12 for trapping on the stage 17, and the beam B13 accurately hits the minute object 10 such as the captured cells. Aiming with a very weak beam, and then increasing the beam intensity
0 is ejected from the capture by the laser beams B11 and B12, and is carried on the laser beam B13 for extraction and is transported to the channel of the extraction device 18 or the tip of the column. The minute object 10 sent out in this way is adsorbed to the channel of the take-out device 18 or the tip of the column and taken out to the outside, where necessary separation and isolation are performed.

A control device (not shown) connected to the trap laser beam projecting means and the take-out laser beam projecting means controls the laser beam projected therein, and particularly, by the take-out laser beam B13. In order to eject the minute object 10, the position and the intensity of the beam B13 are controlled for the beam projection means.

In the cell-manipulating / sorting / picking-up apparatus having the above-mentioned structure, when the minute objects 10 such as cells and intracellular organelles are sequentially drawn and captured between the opposing laser beams B11 and B12, those minute objects are captured. 10 is pushed in the direction of travel of the respective laser beams B11, B12, but since both beams B11, B12 are projected facing each other, they are held in line between the respective focal points made by both laser beams. Then, these minute objects 10 can be ejected one by one in the direction of the beam B13 by projecting a laser beam B13 for extraction, and can be separately collected one by one by a channel or column arranged ahead of them. You can

The following method can be used to capture a minute object with the opposing laser beams. The first is a method of waiting for a minute object to enter between the laser beams. The present inventor has succeeded in the capture experiment by this method. When the capture is performed by this method, the beam can be always turned on, but the beam can also be turned on when a target cell or the like comes on the laser beam. The second is a method of moving a laser beam in parallel to chase cells and the like. Also at this time, there is a method of exposing the particles which are in the middle of sweeping while keeping the beam turned on, and a method of turning on when the particles of interest come on the beam. The beam may be moved by moving the light source of the beam or by moving the sample container with the beam fixed. The third method is a method of pouring cells into the beam, and these methods can be appropriately selected and used.

FIG. 2 shows a second embodiment of the minute object processing apparatus of the present invention.
An example is shown. In the second example, a large number of intersecting guideways projecting a laser beam are used to capture, sort, and isolate microscopic objects such as cells and intracellular organelles. The configuration of the multi-beam type cell sorting and collecting apparatus as described above is shown.

In the multi-beam type cell sorting and collecting apparatus of the second embodiment, as can be seen from FIG.
Laser beams B21 to B23 along a plurality of guideways 21 to 23 for moving 0, and laser beams B24 to B along a plurality of other guideways 24 to 26.
26 and 26 are provided with a laser beam projection means (not shown) for the guide way, which is arranged so as to cross each other and project them. Further, a final take-out device 28 such as a micropipette is provided on the exit side of the guideway.

A laser device is individually used as a means for projecting the above-mentioned laser beams B21 to B26, and these laser devices respectively guide the guideways 21 to 26 diagonally intersecting on the stage 27 of the observation device. A laser beam is projected along it. Then, by this laser beam, the minute object 20 such as a cell in the guideway is delivered to the exit side based on the principle described later.

A control device (not shown) connected to the laser beam projecting means independently controls the intensity of each laser beam projected on them.
In particular, control is performed to move a minute object along the intended guideway side laser beam at the intersection of the guideways.

Observation points are set in front of the intersections of the guideways, and observation devices are installed at these observation points. Based on the output, the observation device mutually changes the intensities of the beams passing through the intersecting guideways through the control device, and controls the minute object 20 to move along one of the guideways. It is a thing. Therefore, if the control device is programmed so that a minute object such as a target cell comes to the outlet of a specific guideway by a specific laser beam, it is taken out to the outside by a channel or column in the extraction device 28 at the outlet. As a result, a plurality of cells can be sorted, isolated, and collected.

In the multi-beam type cell sorting and collecting apparatus having the above structure, cells, intracellular organelles, etc. are moved by being placed on a laser beam along a number of guideways intersecting with each other. By controlling the intensity of the laser beam, the guideways on which cells, organelles, etc. ride are switched. This allows
One by one or any number of cells, subcellular organelles,
It can be moved to the outlet of any guideway and taken out using a column or the like.

In order to bring minute objects such as cells to be sorted and collected to the start point of the guideway, a method of pouring a solution mixed with cells into the start point 27a and a first laser in the solution mixed with cells are used. There is a method in which the beam is passed through and particles drawn into the beam enter the guideway.

At the observation point before the intersection of the beams,
For example, by observing the shape, size, etc. under a microscope, or by performing spectroscopy, the cells are characterized.The rounded cells are on the right, the long and slender are on the left, the large are on the right, and the small are By allocating cells one after another, such as is on the left, red is on the right, and blue is on the left.
Distribute several types of cells to the exit of each guideway. They can be adsorbed to the tip of the column installed at the outlet and taken out. Therefore, according to the above-mentioned cell sorting and collecting apparatus, cells / intracellular organelles are captured,
Several types of cells can be sorted out and taken out.

In the processing apparatus of the first and second embodiments described above, since cells and the like are captured and taken out by the laser beam, it is not necessary to move the micropipette forward and backward as in the conventional apparatus. It is possible to easily sort and collect minute objects such as cells and organelles. Further, the object to be sorted and collected is not limited to cells and subcellular organelles, and it can be used to separately collect and process fine particles in water, air, and vacuum.

The transportation of particles by the laser beam and the particle trap described above are performed based on the following principle. That is, the force that a laser beam gives to a particle includes the force that occurs due to the transfer of momentum due to refraction when light passes through the particle, which confine the particle on the axis of the laser beam and It acts as a pushing force in the axial direction of the beam. Further, when the two beams are coaxially opposed to each other and irradiated, the particles are pushed from the front and the back to be three-dimensionally captured and fixed.

More specifically, as shown in FIG. 3, when the laser beam 1 bends on the surface of a spherical particle 2, the particle has forces FDi1 and FD due to the transfer of momentum.
Receive i2, FDo1 and FDo2. At this time, if the integrals of the forces in the respective hemispheres are F1 and F2, the sum of these becomes the force applied to the entire particle.

These forces F1 and F2 are applied to the laser beam 1
In the radial direction of the laser beam, when the center of the particle deviates from the axis of the laser beam, the hemispherical part near the axis of the laser beam is in a strong electric field. Therefore, the force acting on the portion of the particle 2 close to the axis becomes strong, and the particle 2 is drawn toward the center of the laser beam. Then, when the center of the particle 2 is on the axis of the laser beam, the forces in the radial direction are balanced,
That is the stable point. On the other hand, in the axial direction of the laser beam, the force FF of the resultant force of F1 and F2 in the axial direction of the laser beam acts on the particle 2.

When the laser beam 1 is a parallel laser beam or a laser beam focused at an acute angle,
The force FF is directed in the traveling direction of the laser beam and produces a force that pushes the particles 2 forward. Therefore, the particles are pushed in the traveling direction of the laser beam. Thus, with a single laser beam, the particle force FF acts, so
In the dimensional trap, it is necessary to hit two laser beams facing each other, and the particles are trapped at a point where the forces FF by the laser beams are balanced. In order to identify the trapped position, it is effective to focus the laser beam at an acute angle, whereby the particles are stably trapped between the respective focal points of the two laser beams.

[0027]

According to the multi-beam type micro object processing apparatus of the present invention described in detail above, it is not necessary to move the micro pipette back and forth as in the conventional apparatus, and micro objects such as cells and intracellular organelles can be processed. Capable of capturing, sorting, and collecting by simple operation.

[Brief description of drawings]

FIG. 1 is a configuration diagram showing a basic configuration of a first embodiment of the present invention.

FIG. 2 is a configuration diagram showing a basic configuration of a second embodiment of the present invention.

FIG. 3 is an explanatory diagram for explaining a principle for holding a minute object based on the present invention.

[Explanation of symbols]

10, 20 Small objects, 21-26
Guide way, B11, B12, B13, B21-B26 laser beam.

Claims (2)

[Claims] 1. A pair of trapping laser beam projecting means for projecting opposed laser beams for capturing a minute object, and a laser beam for ejecting a minute object captured by these laser beams. A multi-beam type micro beam characterized by comprising: a take-out laser beam projecting means for projecting from a direction intersecting the beam; and a control device for controlling the take-out laser beam for ejecting a micro object. Object processing device. 2. A plurality of guideway laser beam projecting means for projecting a laser beam along guideways of minute objects intersecting each other, and controlling the respective laser beam intensities so as to be minute at the intersections of the guideways. A multi-beam type micro object processing device, comprising: a control device that moves an object along a laser beam on a desired guideway side.