JPH06113815A - Container for laser irradiation - Google Patents

Abstract

PURPOSE:To provide a container for laser irradiation, necessitating only a small amount of precious gene, enabling easy operation and free from the problem of contamination with saprophyte, etc., from exterior environment. CONSTITUTION:The objective container is composed of a container 1 having a hole opened at a part of its bottom and a lid 3 having a height lower than that of the container 1. The hole of the container 1 is sealed with a glass plate, plastic plate, etc., having a size comparable to that of the bottom of the container 1 and a thickness of about 0.2mm to enable the focusing with an objective lens 8 of a microscope.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a container for culturing living cells and the like, and more particularly to a laser irradiation container suitable for irradiating living cells in the container with laser light.

[0002]

2. Description of the Related Art The point required for a container for irradiating living cells with laser light focused by an objective lens is that the distance between the living cells of the sample and the tip of the microscope objective lens on the sample side is focused by the objective lens. It is possible to be about 0.2 mm, it is a plane that does not adversely affect the optical characteristics of laser light,
And that the living cells are sealed so as not to be contaminated by various bacteria. A laser irradiation container satisfying these requirements, for an upright microscope in which the container is located under the objective lens, is shown in FIG. 4 (Applied Physics B, 35, 282).
4). On the other hand, nothing has been described in detail for an inverted microscope in which the container is located above the objective lens.

[0003]

DISCLOSURE OF THE INVENTION One of the purposes of irradiating a laser beam is to transfer a gene to a living cell or the like. In this case, it is premised that the living cells are soaked in a medium in which the gene is dissolved, and since the amount of the solution of the gene used in the above conventional technique is large, a large amount of valuable gene is required. Further, when the living cells are adherent animal cells, a cover glass is put in a Petri dish or the like in advance for adherent culture, but the operation of attaching the cultured cover glass to the container is complicated. In addition, the contact surface between the cover glass and the container is in intimate contact via grease, etc., but this intimate contact is caused by the cover glass being pressed, which may cause instability such as displacement of the cover glass. There was a possibility of contamination by various bacteria.

An object of the present invention is to provide a laser irradiation container in which the amount of valuable genes used is reduced, the operation is facilitated, the container itself is stable, and the contamination from the outside can be completely shielded. is there.

[0005]

[Means for Solving the Problems] The above-mentioned object is to make a hole in a part of the bottom surface of a container, and make glass or plastic with a plate thickness of about 0.2 mm that is the same size as the bottom surface of the container and can be focused by a microscope objective lens. This is achieved by using a container to which the above are adhered and a container for laser irradiation composed of a lid lower than the height of the container.

[0006]

The container of the present invention is small and can be arranged in large numbers in a commercially available petri dish for culture. Therefore, by culturing the lid of this container in a petri dish for culturing and covering the lid of the petri dish for culturing, it is possible to carry out culturing in a normal cell culture device. After culturing, cover the container,
Since the space between the lid and the container is sealed with parafilm or the like and laser irradiation can be performed as it is, the operation is not complicated, and since the container itself is an integral body, there is no instability factor. Furthermore, since it is sealed with parafilm or the like, there is no contamination from the outside. Next, regarding the amount of gene used, in the case of a laser irradiation container for an upright microscope, the hole diameter is about 4.0 mm,
If the bottom plate thickness is set to about 1 mm, the gene solution is held by surface tension and laser irradiation is possible even when the bottom surface of the container is on the objective lens side, that is, the container is turned upside down. At this time, the amount of gene solution retained is about 30 μl, and the amount of gene is greatly reduced.

On the other hand, in the case of an inverted microscope in which the objective lens is located below the sample, the amount of gene solution can be arbitrarily changed by the hole diameter or the container diameter. That is, the amount of the gene solution is reduced for both the upright microscope and the inverted microscope.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to FIGS. 1, 2 and 3. In FIG. 1, reference numeral 1 denotes a container, and a cover glass 2 having substantially the same size as the bottom surface is adhered so as to make a hole in the bottom surface and close the hole. 3 is the lid of the container 1,
After covering the container 1, it is sealed with parafilm 4 and placed on the stage 5 of the microscope. The cells 6 in the container 1 have been cultured in advance, are immersed in a culture solution 7 containing genes and the like, and are irradiated with a laser beam 9 focused by an objective lens 8. The height of the lid 3 is about half the height of the main body of the container 1, but this prevents the parafilm 4 from hanging on the bottom surface and the bottom surface of the container 1 from being displaced from the vertical plane with respect to the optical axis of the objective lens 8. This is because.

Hereinafter, using the laser irradiation container of the present invention,
Taking the case of irradiating the adherent animal cells with a laser as an example, FIG.
Will be described.

Numeral 10 is a commercially available plastic dish for culture, which can accommodate a large number of laser irradiation containers of the present invention having an outer diameter of about 25 mm. When the lid of the container for laser irradiation is removed and the lid of the plastic dish 10 is covered, there is a gap A between the lid of the plastic dish 10 and the dish main body, so that the culture can be carried out in an ordinary incubator of carbon dioxide gas atmosphere. After culturing the cover glass on the surface of the cover glass, the culture medium is dipped in a predetermined gene solution or the like, covered with a lid 3 as shown in FIG. In the case of the inverted microscope of FIG. 1, the amount of gene solution or the like may be as small as shown in the figure.
As a device for irradiating a laser, there is a device for irradiating a pulsed laser by opening a laser shutter and scanning a stage of a microscope in a rectangular shape. In this case, the sample is always focused on the order of μm with respect to the scanning of the stage, that is, with respect to the cell surface in the laser irradiation range,
The optical axis of the objective lens 8 is required to be exactly vertical. Since the diameter of the cover glass 2 is almost the same as the outer shape of the container 1, the container 1 is always held on the stage 5 of the microscope by the surface of the cover glass 2, and the inner and outer surfaces of the container 1 to which cells are attached are covered with the cover glass 2. Since the parallel plane is maintained exactly according to the criterion of 2, the stage 5 and the cell plane are exactly parallel. On the other hand, since the stage 5 has an accurate vertical plane with respect to the optical axis of the objective lens 8, the optical axis of the objective lens 8 is accurately vertical with respect to the cell surface. That is, the surface of the cell is always focused on the scanning of the stage 5.

The above is the case of using the inverted microscope, but when using the upright microscope in which the objective lens 8 is located on the upper side of the sample, the hole diameter is about 4.0 mm and the plate thickness of the bottom surface is about 1 mm. When the liquid volume is set to a small amount as shown in FIG. 2, the gene solution is retained by surface tension and laser irradiation is possible even when the bottom surface of the container 1 is on the objective lens 8 side, that is, the container 1 is turned upside down. . The amount of gene solution retained at this time is
It is about 30 μl. According to the above examples, a large amount of valuable genes, which was a drawback of the prior art, was required, operations were complicated, and the structure of the container was unstable, and there was a possibility of contamination from external bacteria. There is an improvement.

[0012]

EFFECTS OF THE INVENTION According to the present invention, since a small amount of valuable genes is required and the cells can be directly cultivated in the laser irradiation container, the operation at the preparation stage of the sample can be simplified, and the laser shutter can be used during laser irradiation. The automatic unmanned operation that opens and scans the stage of the microscope in a rectangular shape and irradiates becomes possible. Further, there is no instability factor in the structure of the container, and there is no possibility of being contaminated by foreign bacteria.

[Brief description of drawings]

FIG. 1 is a cross-sectional view when the present invention is applied to a laser irradiation device using an inverted microscope.

FIG. 2 is a cross-sectional view when the present invention is applied to a laser irradiation device using an upright microscope.

FIG. 3 is a cross-sectional view when culturing using the present invention.

FIG. 4 is a cross-sectional view of a conventional technique.

[Explanation of symbols]

1 ... Container, 2 ... Cover glass, 3 ... Lid, 4 ... Parafilm.

Claims (1)

[Claims] 1. A container in which a hole is made in a part of the bottom surface of the container and glass or plastic or the like having a thickness of about 0.2 mm and having the same size as the bottom surface of the container and which can be focused by a microscope objective lens is adhered, A container for laser irradiation, characterized by comprising a lid lower than the height of the laser.