JPH05181068A - Flat plate for meniscus control - Google Patents

Abstract

PURPOSE:To enable the easy prevention of the intrusion of foreign matter into a culture soln. in a culture vessel with good accuracy in a wide field of view and the microscopic observation thereof without disturbing the inside of the culture soln. CONSTITUTION:A transparent flat plate 1 to be floated in the soln. 71, for example, culture soln., in the vessel, such as cell culture vessel, has a flat area sufficient for flattening at least a part of the meniscus of the soln. 71. A part or the whole of the flat plate 1 is preferably hydrophobic. The flat plate 1 is made of, for example, plastic, glass or ceramics and the plane shape thereof is preferably circular or polygonal.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a meniscus control flat plate. More specifically, the present invention relates to a meniscus control plate used for observing a specimen such as a biological tissue or cell after culturing the specimen with a microscope.

[0002]

2. Description of the Related Art Cell culture is widely used in the fields of medicine and biology. In addition, as containers for culture, containers of various sizes are sold, and it has become possible to carry out cell culture using containers suitable for the purpose.

However, the inner diameter of the container is 16.5 mm or less,
In other words, cell culture vessels with more than 24 holes (eg Bexon
Tekkinson Micro Test Plate 3070.9
6-well, multi-well plate 3047 / 24-hole coster, cluster dish 3548/48 well etc.) When trying to observe the cells with an optical microscope, the liquid surface of the culture solution in the container is curved due to surface tension, The image is deteriorated by the lens effect due to. In particular, when observing with a phase contrast microscope, the ring opening 81 in the illumination light must be projected onto the ring coat of the phase difference modulation plate 84 in the objective lens 83 as shown in FIG. Due to the effect, the image is formed at a position and magnification different from that on the ring coat, so that proper phase difference modulation cannot be performed, the image is considerably deteriorated, and the image can be seen only in the central portion of the visual field. In addition, when attempting to measure the cells, the accuracy of the measurement deteriorates due to the curvature of the liquid surface.

[0004]

Therefore, as a method for solving such a problem, there is known a method of dipping an optical member in a solution as disclosed in Japanese Patent Publication No. 2-30469. However, in this case, since the optical member such as a lens is made of an expensive member, it must be repeatedly used. As a result, when the optical member is inserted into a large number of holes containing a solution, there is a risk that foreign matter may be mixed into the culture solution.
In addition, cleaning the optical member is not easy.

Apart from this, a method of inserting a hydrophobic cylinder into the well of a cell culture plate is known (Japanese Patent Laid-Open No. 62-69979). However, this method narrows the observable field of view due to the cylinder, and is difficult to handle when the amount of liquid is small. Furthermore, when the cylinder is inserted into the culture solution, the liquid surface of the culture solution rises, and the whole solution is vigorously agitated. As a result, there is a possibility that specimens such as living tissues or cells cultured on the bottom surface of the culture container may be overstimulated to inhibit the culture, or may be separated from the bottom surface of the container and mixed in the culture solution. Therefore, it is difficult to accurately observe a specimen in a normal culture state using the cylinder.

Therefore, an object of the present invention is to observe a culture solution in a culture vessel with a wide field of view, with high precision, to easily prevent foreign matter from being mixed into the culture solution, and to observe with a microscope without disturbing the inside of the culture solution. It is an object of the present invention to provide a device for controlling the meniscus of a liquid surface such as a culture solution that enables the above.

[0007]

The present invention is a transparent flat plate that floats on a solution in a container and has a plane area sufficient to flatten at least a part of the meniscus of the solution. Regarding flat plates. The present invention will be described below.

In the present invention, the container is, for example, a cell culture container. A specific example is a multiwell having a cylindrical well of 24, 48 or 96 holes. However, there is no particular limitation as long as the inside of the container is directly observed with a microscope. More specifically, the container of the present invention is a well of a plate for culturing living tissues or cells having an inner diameter of about 5 to 25 mm. The smaller the inner diameter of the container, the greater the adverse effect on the microscopic observation of the meniscus, and the use of the flat plate of the present invention is effective.

The flat plate of the present invention can be made of, for example, plastic, glass or ceramics. However, the material is not particularly limited as long as it is a transparent flat plate. still,
It is preferable to use a plastic that has high transparency, has a low specific gravity, and can be easily floated in a solution. An example of plastic is polymethylmethacrylate (PMMA) (specific gravity:
1.08 to 1.20), polystyrene (specific gravity: 1.04)
~ 1.11), polyethylene (specific gravity: 0.910-0.
965), polycarbonate (specific gravity: 1.20), polyester (specific gravity: 1.01 to 1.46), vinyl chloride (specific gravity: 1.35 to 1.45) and the like.

It is preferable that part or all of the surface of the flat plate is hydrophobic, from the viewpoint that the flat plate can be easily floated in the solution. Further, since the surface is hydrophobic, it is possible to prevent the solution from flowing around the upper surface of the flat surface and wetting the upper surface. Examples of the method for making a flat plate hydrophobic include a method of coating a flat plate with a hydrophobic substance (polymer) to form a film, a chemical reaction of a hydrophobic compound, a plasma reaction, a reaction using an electron beam, radiation or ultraviolet rays. The method of bonding on a flat plate, a CVD (chemical vapor deposition) method, etc. can be mentioned.

In addition to hydrophobizing the surface of the flat plate, the flat plate can be made of a substance (for example, Teflon) having a low surface energy and difficult to be wet with water or a solution. The degree of hydrophobicity is measured by a droplet method in which a water droplet is placed on a flat plate and the contact angle is read.
A bubble method of attaching air bubbles in water, a hanging plate method of hanging a flat plate, or the like can be used. The contact angle is, for example, preferably 30 ° or more in the case of the droplet method, and more preferably 60 ° or more. For example, in the case of PMMA, it is 64 °.

It is preferable that the flat plate surface is sterilized from the viewpoint of preventing contamination in the culture solution. Examples of the sterilization method include a method of heat treatment in an autoclave, a method of sterilization with ethylene oxide (EO) gas, and a method of irradiating with radiation such as ultraviolet rays, depending on the material of the flat plate.

The flat plate 1 of the present invention flattens at least a part of the meniscus 70 formed on the surface of a solution 71 such as a culture solution in a container 72 as shown in FIG. 2 as shown in FIG. is there. The meniscus 70 may have a convex shape other than the concave shape as shown in FIG. 2 depending on the surface energy of the solution 71 and the surface energy of the inner wall 73 of the container 72. The flat plate of the present invention is effective for both concave and convex meniscuses. The area of the liquid surface of the solution flattened by the flat plate (that is, approximately equivalent to the flat area of the flat plate) is
The wider the width, the wider the visual field for microscopic observation, which is preferable. If the inner diameter of the container (well of the culture plate) is small, it is preferable that the flat area of the flat plate is almost equal to the horizontal cross-sectional area in the container and almost covers the surface of the solution. On the other hand, for a container with a relatively large inner diameter,
In some cases, it is not necessary to cover the entire surface of the solution. Generally, it is appropriate that the surface area of the flat plate is 50% or more of the horizontal cross-sectional area in the container from the viewpoint of ensuring a wide field of view.

There are no particular restrictions on the planar shape and dimensions (thickness) of the flat plate. As a plane shape, a circle (a perfect circle or an ellipse) is generally used, but a polygon may be used. Regarding the thickness, considering the mechanical strength of the flat plate, the workability, and the fact that it floats in a solution, for example, 0.3 to 2 mm, preferably 0.5 to 1 mm
Is appropriate.

The flat plate of the present invention will be described with reference to FIGS. A perspective view of the meniscus control flat plate 1 of the present invention is shown in FIG. The meniscus control flat plate 1 is made of polymethylmethacrylate (hereinafter referred to as PPM. Specific gravity: 1.188).
[25 ° C.]), and the upper surface 11 and the lower surface 12 thereof are parallel planes. The thickness of this flat plate 1 is 0.5
The upper surface 11 and the lower surface 12 have a diameter of 10.5 mm and are suitable for use in a culture container having an inner diameter of 11 mm.

The meniscus control flat plate 2 shown in FIG. 6 is formed by forming a grip portion for gripping the flat plate by a gripping tool on the side edge 21 of the disc shown in FIG.
2 and 22 ^′ are formed as semicircular plan view diameter 1.5 mm cuts from the side peripheral edge 21 of the flat plate 2 toward the flat plate center 23. This semi-circular cut is the center of the flat plate 2
When the pair of notches 22 and 22 ^' are formed symmetrically with respect to 3 and are grasped by the grasping tool, they cooperate to allow the user to handle them at will. The diameter of this semicircular cut is preferably as small as possible in order to widen the field of view of the speculum, if gripping is possible.

The semicircular notch in plan view of this example is
Not only a semicircle, but the longer arc in plan view (long arc)
It may have a shape or a shorter arc shape (short arc shape).

The meniscus control flat plate 3 shown in FIG. 7 has grip portions 32 and 32 ^′ formed on the side edge 31 of the flat plate 3 in a shape cut out by three sides of a quadrangle in a plan view. The shape of is changed. The length of one side of the quadrangle of the cut portion is 1 mm.

The meniscus control flat plate 4 shown in FIG. 8 is formed by forming gripping portions 42, 42 ^′ on the side peripheral edge 41 of the flat plate 4 in a shape cut by two sides of a triangle in plan view. The shape of the cut is changed. The length of one side of the notch triangle is 1 mm.

In the meniscus control flat plate 5 shown in FIG. 9, a symmetrical position with respect to the center 53 of the disc on the disc-side peripheral edge 51 is viewed in a plane perpendicular to the upper and lower faces of the disc by two parallel planes. A grip portion 52 having a chordal shape,
52 ^' is formed. The plane from which the side edge is cut is located at a position 1 mm from the disk side edge in the plan view in the center direction of the disk.

FIG. 10 shows a side view of the meniscus control flat plate 6. The flat plate 6 has a protrusion 62 on one surface for holding the flat plate 6 when the flat plate 6 moves. The protrusion 62 is the flat plate 6
It is preferable to provide it at a position closer to the periphery than the central part of the plane from the viewpoint of not hindering the visual field.

The flat plate of the present invention is obtained by cutting a round bar manufactured in advance to a predetermined thickness, molding a raw material (solution) into a mold, or polishing the surface of the flat plate obtained by these methods. It can be obtained by a method or the like.

The flat plate is floated on the solution surface by using a suction dropper or tweezers. In the case of using tweezers, a flat plate with a cutout is preferable (see, for example, FIGS. 6 to 9).

In addition, from the viewpoint of use in wells for culture,
Prior to use, it is preferable to sterilize in advance by treatment with an autoclave, ethylene oxide gas, or radiation. In that case, a flat plate is created using a material that can withstand any of these treatments.

The operation of the meniscus controlling flat plate of the present invention will be described below. Normally, as shown in FIG. 2, the surface of the solution 71 placed in the container 72 is not flat and the meniscus 70 is not flat.
To form. When observing the inside of the solution with an inverted phase contrast microscope, if the meniscus 70 exists as shown in FIG. 1, the light 90 transmitted through the solution 71 from the phase difference ring opening 81 on the condenser side through the condenser lens 82 is the objective. Proper phase difference modulation cannot be performed even through the lens 83, and an image is not formed on the phase difference modulating plate 84, so that the condenser aperture image 91 is formed at a position displaced from the modulating plate 84. As a result, the image deteriorates considerably and becomes difficult to observe.

On the other hand, when the flat plate of the present invention is used, the meniscus 70 of the solution 71 in the container 72 is flattened by the flat plate 1 except for the limited portion between the flat plate 1 and the inner wall 73 of the container, as shown in FIG. To be done. As a result, as shown in FIG. 4, the condenser aperture image 92 is formed on the phase difference modulation plate 84.

Usually, for example, in a cylindrical well having an inner diameter of 15 mm, the cylindrical cross-sectional area of about 3 is obtained without using the flat plate of the present invention.
For example, the diameter is 13 while the field of view is only 0%.
When a flat plate having a size of mm (the flat area of the flat plate is 95% of the cylindrical cross-sectional area) is used, a visual field of about 85% or more can be obtained.

[0028]

According to the meniscus controlling flat plate of the present invention, it is caused by the meniscus formed on the surface of the culture solution in the well when microscopically observing living tissues and cells cultured in the well of the culture plate. This is extremely useful because it can improve the narrowing of the microscopic field and allows accurate observation of living tissue and cells in a normal culture state.

Furthermore, the prior art Japanese Patent Laid-Open No. 62-6997.
In the case of the device disclosed in Japanese Patent No. 9, when the liquid is moved in and out of the aqueous solution, the liquid level rises and falls, and accordingly, the entire aqueous solution is fluidly stirred. On the other hand, the meniscus control flat plate of the present invention has almost no change in the liquid surface, and the whole liquid is maintained in a state in which it is almost unchanged from the stationary state,
It also has the advantage that it has no adverse effect on the cultured cells.

Further, the meniscus control flat plate of the present invention is inexpensive, and it is very economical because it can be used with one plate even when observing only one hole without using all the well holes. Furthermore, since it can be used as a disposable flat plate, it is extremely useful for users.

[0031]

EXAMPLES The present invention will be described below with reference to examples. Example 1 A flat plate made of polymethylmethacrylate was cut and polished to obtain a meniscus control flat plate of the present invention having a thickness of 0.5 mm and a flat surface diameter of 10.5 mm, which was EO sterilized.

Separately from this, human lens epithelial cells with anterior capsule pieces obtained at the time of cataract surgery were prepared using a 48-well multiwell for cell culture and 0.5 m of MEM solution containing 10% fetal bovine serum.
1 was added to the well, and static culture was carried out at 37 ° C. in an atmosphere of 5% carbon dioxide and 95% air for 3 weeks. Observation was performed with an inverted phase contrast microscope (Olympus IMT-2) according to a conventional method.
The result is shown in the photograph of FIG. Only the central part (about 25% of the total field of view) is visible, and the entire field of view cannot be observed. The above-mentioned PMMA flat plate slightly smaller than the inner diameter of the well of 11 mm was floated in the culture solution and the same portion was observed. The result is shown in the photograph of FIG. Focusing on the characteristic cells in both photographs, the photograph of FIG. 13 showed a clear image in the entire visual field (nearly 100%).

Example 2 The same operation as in Example 1 was repeated except that the diameter was 10.5 mm.
Fig. 14 shows the result when a flat plate of mm was created and used.
Shown in the photo. It can be seen that cells can be clearly observed under the flat plate, but they can hardly be observed outside the flat plate, and that clear observation can be made by using the flat plate.

Example 3 The same 10.5 mm flat plate as in Example 1 was used, and the same operation was performed to observe with a 20 × objective lens. The result is shown in the photograph of FIG. An extremely clear image was obtained, enabling detailed morphological observation of cells. From this photograph, it was found that the cells were present on the anterior capsule, the anterior capsule and the bottom of the well, and each exhibited a characteristic morphology.

Example 4 By the same operation as in Example 1, 15.5 mm and thickness 1 mm
Make a flat plate of this and use this for 24-well multiwell (inside diameter 1
6.5 mm). As a result, a clear visual field could be obtained as in Example 1.

Example 5 Observation was carried out in the same manner as in Example 1 except that an upright microscope was used instead of the inverted phase contrast microscope.

Conventionally, when the culture container was illuminated from the bottom and observed with an upright microscope which can be observed from the top of the container, it was impossible to observe due to the curved surface of the culture solution. However, using the flat plate of the present invention, by flattening the meniscus of the solution in the culture vessel, a bright field by an upright microscope, phase difference,
It became possible to observe Nomarski and others. 11
As shown in FIG. 6, by floating the flat plate 1 of the present invention in the solution 71, the condenser aperture image 92 can be formed on the phase difference modulation plate 84.

[Brief description of drawings]

FIG. 1 is an explanatory diagram for observing a solution in a container with an inverted phase contrast microscope.

FIG. 2 is a cross-sectional view showing a meniscus of a solution in a container.

FIG. 3 is a cross-sectional view showing a flat plate 1 of the present invention floated on a solution in a container to be flattened.

FIG. 4 is an explanatory diagram for observing a solution in which the flat plate 1 of the present invention is floated with an inverted phase contrast microscope.

FIG. 5 is a perspective view of the flat plate 1 of the present invention.

FIG. 6 is a plan view of the flat plate 2 of the present invention.

FIG. 7 is a plan view of the flat plate 3 of the present invention.

FIG. 8 is a plan view of the flat plate 4 of the present invention.

FIG. 9 is a plan view of the flat plate 5 of the present invention.

FIG. 10 is a side view of the flat plate 6 of the present invention.

FIG. 11 is an explanatory diagram for observing a solution in which the flat plate 1 of the present invention is floated with an upright microscope.

FIG. 12 is a photograph instead of a drawing showing the morphology of living things.

FIG. 13 is a photograph instead of a drawing showing the morphology of living things.

FIG. 14 is a photograph instead of a drawing showing the morphology of living things.

FIG. 15 is a photograph instead of a drawing, which shows the morphology of organisms.

[Explanation of symbols]

1, 2, 3, 4, 5, 6, 6: Meniscus control flat plate of the present invention 70: Meniscus

Front page continued (72) Inventor Yoshihiro Kono 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Co., Ltd.

Claims (6)

[Claims] 1. A flat plate for meniscus control, which is a transparent flat plate floatable on a solution in a container and has a flat area sufficient to flatten at least a part of the meniscus of the solution. 2. The flat plate according to claim 1, wherein a part or all of the flat plate surface is hydrophobic. 3. The plate according to claim 1, wherein the container is a cell culture container. 4. The flat area of the flat plate is 5 of the horizontal sectional area in the container.
The flat plate according to claim 1, which is 0% or more. 5. The flat plate according to claim 1, which is made of plastic, glass or ceramics. 6. The flat plate according to claim 1, wherein the flat plate has a planar shape of a circle or a polygon.