JPH04356183A - Light pressure-type cell sorter - Google Patents

Abstract

PURPOSE:To select cells in the same liquid without damaging cells by controlling two laser beams and XY stages of two laser irradiation devices by using a computer. CONSTITUTION:A cell storage container 30 is divided into two chambers 34a and 34b by a low partition wall 32 at the central part. The base 36 of one chamber 34a is made into a beam irradiation window to which a laser beam 46 in the vertical direction is introduced and a side wall 38 is processed into a beam irradiation window to which a laser beam 50 in the horizontal direction is sent. The laser beam 50 has a higher intensity than the laser beam 46. A first XY stage 48 is equipped with a laser irradiation hole 40 and a condensation lens 44 and a second XY stage 56 is provided with a laser irradiation hole 52 and a condensing lens 54 to focus the laser beam. The action of the XY stages 48 and 56 are controlled while maintaining the Y co-ordinates in the same coordinate by a computer 60.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell sorter used for sorting target cells in the field of biotechnology.

0002

2. Description of the Related Art Cell sorters include cell sorters that combine sheath flow and electric field separation, and cell sorters that use a pick-up method using a micromanipulator. The former cell sorter is configured as shown in Fig. 2 and is put into practical use under the name of flow cytometer or cell sorter (Metabolism magazine, VOL. 21, special issue ``Immunology 84'', pp. 189-198). (See Nakayama Shoten, 1984).

In FIG. 2, the cell suspension 4 is positioned at the center of the nozzle 2, and the cells are made to pass precisely through the center of the nozzle 2 by flowing a water sheath 6 outside the cell suspension 4. There is. By vibrating the nozzle 2 with the piezoelectric crystal 8, the cell flow 3 from the tip of the nozzle 2 is transformed into water droplets 3.
Make the position a uniform. A laser beam from the laser device 10 is focused onto the cell stream 3 by a lens 12 . Fluorescent light emitted from cells by laser beam irradiation is collected by a lens 14 in a direction perpendicular to the laser beam, and separated by a dichroic mirror 16 according to the wavelength of the fluorescence. One of the branched fluorescent lights is detected by the first photomultiplier tube 18, and the other fluorescent light is detected by the second photomultiplier tube 20.

On the other hand, the light scattered by the laser beam by the cells is detected as forward scattered light by the optical scanner detection tube 22, and output as a signal corresponding to the size of the cell. Cells in the cell stream 3 are identified by the detection signals F1, F2 from the photomultiplier tubes 18, 20 and the signal S from the optical scanner detection tube 22. Based on these signals F1, F2, and S, it is determined whether to apply a voltage of +100V or -100V to the water sheath 6, or whether to apply no voltage. When a voltage is applied to the water sheath 6, the water droplet 3a becomes electrically charged. A deflection plate 24 is provided at a position below the tip of the nozzle 2.
A voltage of 000V is applied. The water droplets 3a containing cells that are dropped from the tip of the nozzle 2 are collected by changing the direction of the droplets 3a by a deflection plate 24 depending on whether they are charged or not and whether they are positive or negative.

[0005]

[Problem to be solved by the invention] In the cell sorter shown in Figure 2, high pressure is applied during cell transport, so cells that are susceptible to mechanical shock, such as protoplasts, are destroyed when the cells are ejected into the atmosphere from the nozzle. . Moreover, it is not possible to separate large cells such as fertilized eggs or small cells such as microorganisms, which have cell diameters outside the range of 20 to 100 μm. The cell sorter shown in Figure 2 has a complicated device and is large in size.
Becomes expensive. Additionally, staining with fluorescence is a mandatory condition. The micromanipulator method requires skill and has the disadvantage that the selection speed per unit time is slow. An object of the present invention is to provide a cell sorter that has a simple configuration and is capable of sorting target cells without destroying the cells.

[0006]

[Means for Solving the Problems] The cell sorter of the present invention includes:
The room is divided into two rooms by a partition wall, and one of the rooms is provided with a beam irradiation window on the bottom and side surface that allows the laser beam to pass through without significant attenuation. a cell storage container set at a height through which an incident horizontal laser beam can pass; a first laser irradiation device that makes a first laser beam incident vertically from the beam irradiation window on the bottom; and the side surface. a second laser irradiation device that horizontally injects a second laser beam with a higher intensity than the first laser beam from a beam irradiation window of the laser beam; a respective XY stage that moves each of the laser beams in a horizontal plane; 2 X
This is an optical pressure type cell sorter equipped with a positioning control mechanism that controls the two XY stages by keeping the coordinates in the direction parallel to the beam irradiation window on the side surface of the Y stage the same.

[0007]

[Operation] A buffer solution is filled in the cell storage container to a level higher than the partition wall, and cells containing necessary cells and unnecessary cells are placed in one of the rooms provided with the beam irradiation window. After irradiating the target cell with the first laser beam to move the cell upward, the cell is moved horizontally by the second laser beam and moved through the partition into a second chamber, and then the cell is irradiated with the second laser beam. The cell is caused to naturally fall into the other room by stopping the emission of light or by moving the second laser beam. For example, it is known that cells move due to the optical pressure of a laser beam.
(2013), pages 417-422.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 schematically represents one embodiment. 30 is a cell storage container, which is divided into two chambers 34a and 34b by a partition wall 32 in the center. The partition wall 32 is low and set at a height that allows the second laser beam 50 to pass over the partition wall 32. One room 34a
The bottom surface 36 serves as a beam irradiation window through which the first laser beam 46 enters in the Z-axis direction (vertical direction), and one side wall 38 serves as a beam irradiation window through which the second laser beam 50 enters in the horizontal direction. It has become. A first laser irradiation port 40 that irradiates a laser beam in the Z-axis direction is provided below the first chamber 34a, and a first laser oscillator 42
A laser beam from is guided to a laser irradiation port 40 by an optical fiber cable. 44 is an aperture lens that converges the laser beam 46 in the Z-axis direction. The laser irradiation aperture 40 and the aperture lens 44 are attached to a first XY stage 48 and can be moved in the XY direction (in the horizontal plane). The first laser beam 46 is transmitted through the beam irradiation window 36
The light enters the first chamber 34a in the Z-axis direction. A laser irradiation port 52 and an aperture lens 54 for converging the laser beam are attached to the second XY stage 56 in order to cause the horizontal laser beam 50 to be incident from the beam irradiation window 38 on the side surface. A laser oscillator 5 is connected to the laser irradiation port 52.
A laser beam is guided from 8 by a fiber optic cable.

The XY stages 48 and 56 are connected to the computer 6
Controlled by 0. XY stages 48 and 56 are arranged so that laser beams 46 and 50 intersect in first chamber 34a.
is controlled while keeping the Y coordinate at the same coordinate. The side wall 62 of the second chamber 34b of the cell storage container 30 is a stop plate against which the cells moved by the horizontal laser beam 50 hit. A buffer solution 63 for storing cells is placed in the cell storage container 30 up to a level higher than the partition wall 32 . The horizontal laser beam 50 is set at a position higher than the partition wall 32 and at a position where it passes through the buffer liquid 63. The intensity of the laser beam 50 is set higher than that of the laser beam 46.

Next, the operation of this embodiment will be explained. A mixture of necessary cells and unnecessary cells is placed in one chamber 34a. Cells 64 indicated by black circles are necessary cells, and cells 66 indicated by white circles are unnecessary cells. The XY stages 48 and 56 are operated to move the optical axis of the laser beam 46 in the Z-axis direction directly below the target cell, and the target cell is irradiated with the laser beam 46 from below. Irradiation with the laser beam 46 lifts the cell 64a in the Z-axis direction (upward). When the cell 64a is lifted up to the intersection P of the two laser beams 46 and 50, the horizontal laser beam 50 has a larger output, so the cell 64a
a is captured by the laser beam 50 at that point (transfer direction displacement point) P, and moves along the laser beam 50 in the direction of the stop plate 62 . The cells extend over the septum 32 into the second chamber 34
When the laser beam 50 stops emitting light after entering b,
The cells naturally fall into the second chamber 34b. Similarly, other necessary cells are transferred from the first chamber 34a to the second chamber 34a.
room 34b.

It is preferable that the diameters of the laser beams 46 and 50 be narrowed down to a size close to the diameter of the cells to be moved. The cells to be treated are generally in the range of 0.5 to 200 μm. The target cells to be sorted are distinguished by their morphology and the presence or absence of coloration. Fluorescent or non-fluorescent vital stains are used for coloring.

When the operator manually applies the laser beam 46 to target cells, the position of the cells is confirmed using a microscope, and the laser beam 46 is moved by operating the XY stage. The present invention can also be combined with a microscopic image processing system to detect the location of cells. When combined with an image processing system, it is possible to create an automated device that automatically performs continuous selection by linking the color and shape determination results to the movement of the laser beam.

[0014]

Effects of the Invention In the present invention, cells are selected in the same liquid without applying mechanical impact, so there is no damage to the cells. By changing the diameter of the laser beam using a diaphragm mechanism, it can be applied to cells of various sizes. It is also possible to select only cells with a special morphology using human judgment or image processing technology. Fluorescent staining, which requires complicated work, is not necessarily required. And the device is simple.

[Brief explanation of drawings]

FIG. 1 is a schematic perspective view showing one embodiment.

FIG. 2 is a schematic diagram showing a conventional cell sorter.

[Explanation of symbols]

30 Cell storage container 32
Partition wall 34a First room 34b
Second room 36, 38 Beam irradiation window 40, 52 Laser irradiation port 42, 58 Laser oscillator 44, 54 Aperture lens 46, 50 Laser beam 48, 56 XY stage

Claims (1)

[Claims] Claim 1: The partition wall divides the room into two rooms, one of the rooms is provided with a beam irradiation window on the bottom and side surfaces thereof through which the laser beam passes without significant attenuation; a cell storage container set at a height such that a horizontal laser beam incident from the beam irradiation window can pass therethrough; and a first laser irradiation device in which a first laser beam is incident vertically from the beam irradiation window on the bottom surface. a second laser irradiation device that horizontally injects a second laser beam with a higher intensity than the first laser beam from the beam irradiation window on the side surface; and a second laser irradiation device that moves each of the laser beams in a horizontal plane. An optical pressure type cell sorter comprising an XY stage and a positioning control mechanism that controls the two XY stages by keeping the coordinates of the two XY stages the same in a direction parallel to the beam irradiation window on the side surface.