JP2003177331A - Culture board for microscope observation and observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture board for microscope observation which can culture an organism specimen and is less deteriorated in imaging performance in observing the organism specimen and to provide an observation method suitable for observation of the organism specimen by using this culture board. <P>SOLUTION: The culture board for microscope observation consists of a transparent fluororesin having a refractive index of ≥1.33 to ≤1.34 and is subjected to treatment for hydrophilicity impartation on the board surface. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a water immersion objective lens, and more particularly to a culture substrate for microscopic observation used when observing a biological specimen using a water immersion objective lens having a high magnification and a high numerical aperture.

[0002]

2. Description of the Related Art In recent years, three-dimensional structural analysis of cytoskeletons and chromosomes with a scanning laser microscope having high contrast and high resolution, localization of intracellular fluorescent substances by fluorescent antibody method using a scanning laser microscope, etc. , Is attracting great attention in the field of basic research in biology and medicine.

A major feature of the scanning laser microscope is that a three-dimensional image is obtained by optically slicing a sample with a confocal optical system. By the way, when using this scanning laser microscope, for example, when observing a biological specimen (such as cells) immersed in a culture solution,
A structure that does not cause deterioration of the imaging performance is required.

As a technique for preventing the deterioration of the image forming performance, the technique disclosed in Japanese Utility Model Laid-Open No. 5-64816 is known. According to this, an objective lens having a refractive index similar to that of the culture solution is required, and in an objective lens having a high magnification and a high numerical aperture, the imaging performance is deteriorated even by a small thickness error of the cover plate. It has been pointed out that the refractive index of the cover plate is about the same as the refractive index of the culture solution.

[0005]

However, there are still problems to be solved when observing a specimen using the above-mentioned technique. The specimen in the culture solution can be observed as a biological specimen by adhering it on a glass called a culture substrate and observing it as a biological specimen, but it is necessary to observe the boundary part by observing the adhesion state between the culture substrate and the biological specimen. It may be said that.

Therefore, the culture substrate on which the sample is loaded is tilted with respect to the optical axis of the objective lens, and the scanning surface of the scanning laser microscope is aligned with the surface to be observed, and the sample and the culture substrate are observed as one body. Although necessary, in this case, a clear image cannot be obtained due to the difference between the refractive index of water (1.333) and the refractive index of the culture substrate glass (1.521) near the boundary between the substrate and cells. There was a problem.

On the other hand, the above-mentioned prior art (Actual Kaihei 5-648)
In 16), a cover plate using Cytop (trade name: transparent fluororesin manufactured by Asahi Glass Co., Ltd., refractive index: 1.33 to 1.34) is used, and the refractive index of water and that of the cover plate are about the same. It is disclosed that by doing so, a clear image can be obtained without being affected by the change in the cover plate thickness and the poor surface accuracy.

Therefore, it is conceivable to use this CYTOP as a culture substrate, but since the water repellency of the CYTOP surface is very high, it is necessary to apply collagen for culturing a specimen to the culture substrate and fix it. Therefore, it was difficult to use Cytop as a culture substrate. Furthermore, because the water repellency of the Cytop surface is very high, the adhesion between the biological specimen and the culture substrate is not sufficient, and there is a restriction in observing from various angles by tilting the culture substrate. Was not applied.

The present invention has been made in view of the above problems, and uses a water-immersion objective lens, in particular, a scanning laser microscope equipped with a high-magnification, high-numerical-aperture water-immersion objective lens. To provide a culture substrate for microscopic observation in which a biological specimen can be cultivated when observing, and the imaging performance is less deteriorated, and an observation method suitable for observing a biological specimen using the culture substrate. With the goal.

[0010]

Means for Solving the Problems The present invention for solving the above-mentioned problems includes a culture for microscopic observation, which is made of a transparent fluororesin having a refractive index of 1.33 or more and 1.34 or less and whose substrate surface is hydrophilized. The substrate.

Further, the present invention is the culture substrate for microscope observation according to the above-mentioned invention, wherein an ultraviolet light source is used for the hydrophilic treatment of the substrate surface.

The present invention also provides the culture substrate for microscope observation according to the invention described above, wherein a part of the surface of the substrate is subjected to hydrophilic treatment.

Here, "a part" of the substrate surface means at least one part of the substrate surface. Therefore, this includes the case where there are hydrophilized portions at a plurality of locations on the front surface of the substrate, and the portions are not limited to one side and include those present on both the front and back sides.

The present invention further provides a method for observing a biological specimen using the culture substrate for microscope observation according to the above-mentioned invention, wherein the culture substrate for microscope observation is arranged at an arbitrary angle with respect to the optical axis of the water immersion objective lens. It is a method of observing a biological specimen to be observed by doing so.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION One of the present invention is to modify a surface of a transparent fluororesin typified by Cytop so that collagen or the like can be applied and the adhesion to a biological specimen can be improved. Is. The inventors have made diligent studies on this point, and in order to enable collagen application,
We have come to the view that we should improve the wettability by modifying the water repellency of CYTOP. Then, based on this viewpoint, the inventors conducted repeated tests on a hydrophilic treatment method for improving the wettability of the surface of Cytop and found a treatment method that can be used as a culture substrate.

FIG. 1 is a schematic flow chart for explaining a method for modifying a culture substrate for microscopic observation according to the present invention.

First, after processing a sample of fluorocarbon resin Cytop into a size that is easy to handle, the fluorocarbon resin is immersed in water (S1), and an ArF excimer laser is irradiated as an ultraviolet light source to modify the sample surface. (S2).

This treatment is known as a hydrophilic treatment for improving the wettability of the fluororesin, and it is a transparent fluororesin having a refractive index of 1.33 or more and 1.34 or less. The processing conditions for modifying the top to a state suitable for a culture substrate have not been clarified. The inventors repeated the test under various conditions, and as a result, as an irradiation condition of the excimer laser for modifying the state suitable for the culture substrate, the energy density was 5 to 100 mJ.
It was found that the ranges of / cm ² , the number of repeated transmissions of 1 to 100 Hz, and the number of shots of 3,000 to 50,000 were appropriate.

In this embodiment, the irradiation conditions are energy density of 20 mJ / cm ² , repetition frequency of 30 Hz, 2
5,200 shots were used. Under this irradiation condition, the number of shots is 2,000 to 3,000 in the usual hydrophilic treatment.
However, the value is about 10 times that. It is believed that in a general hydrophilic treatment, a UV laser is used to introduce a uniform amino group into a specific surface of a fluororesin by a non-thermal photochemical reaction, thereby increasing hydrophilicity. It is considered that in order to modify it for a culture substrate, irradiation is required until the hydrophilic treatment is further advanced and the surface roughness of the fluororesin is changed. still,
The contact angle of water changes from 87 ° before the treatment to 64 ° after the treatment, and the effectiveness of the treatment can be confirmed also from the change in the contact angle.

Following the above hydrophilic treatment, a pretreatment for culturing is performed. The pretreatment for culturing is a general treatment, and the hydrophilically treated culture substrate is sterilized by UV irradiation for 30 minutes (S3) → washed with 70% ethyl alcohol, water, PBS (S4). ) → About 10 minutes albumin immersion (S5) → PBS washing (S6) → 1 /
10 Collagen application (S7) → washing with PBS (S
8) is performed.

In this embodiment, CYTOP is used as the transparent fluororesin, but NEOFLON (trade name: transparent fluororesin manufactured by Daikin Industries, Ltd.) may be used instead of CYTOP. Other organic materials may be used as long as the refractive index is about the same as the refractive index of water (1.333). Also, when an ultraviolet light source is used for the hydrophilic treatment, it is not limited to the ArF excimer laser, and a UV lamp, an excimer lamp or the like can be used depending on the situation. Further, it is also possible to cover the hydrophilic treatment portion applied to a part of the culture substrate with an arbitrary shape by covering it with a mask like a semiconductor manufacturing apparatus when performing the hydrophilic treatment.

Next, a method of observing a biological specimen using the thus prepared culture substrate will be described.

FIG. 2 is a diagram showing a first embodiment of the observation method according to the present invention.

The water immersion objective lens 1 is immersed in water 4 in a container (not shown) with the tip of the lens mounted on a scanning laser microscope (not shown).

The culture substrate 2 is processed into a size (about 15 mm × 5 mm × 0.2 mm) that is easy to handle using Cytop. A part of one side of the culture substrate 2 is subjected to a hydrophilic treatment using an ArF excimer laser as an ultraviolet light source and a pretreatment for culturing, and the cultured cells 3 are attached in a live state. There is. The cell 3 is a HeLa cell, which was cultured at 37 ° C. and 5% CO ₂ and then introduced with a gene for microscopic observation.

The culture substrate 2 is arranged substantially parallel to the optical axis of the water immersion objective lens 1 (that is, the culture substrate 2 and the virtual observation surface 5 are orthogonal to each other), and the portion to be observed is within the visual field. Has been adjusted. In the present observation method, since the culture substrate 2 that has been subjected to the hydrophilization treatment described above is used, the culture substrate 2 can be arranged parallel to the optical axis while keeping the culture substrate 2 and the cells 3 attached to each other. By the operation, it is possible to optically slice the cells 3 and observe not only the cells 3 but also the adhered state of the culture substrate 2 and the cells 3.

FIG. 3 is a diagram showing an image observed by a microscope.

In the image, the culture substrate 2 is arranged on the left side, but the refractive index of the culture substrate 2 is 1.33 or more and 1.34.
Since it is below and extremely close to water, it is possible to optically realize a state in which cultured cells are floating in water while being alive (while being attached to the substrate). The observed image is a clear image with little deterioration in the image forming performance.

Further, since the culture substrate 2 and the cells 3 are attached to each other, it becomes possible to observe the culture substrate 2 while changing the angle with respect to the optical axis as shown in FIG. An image of an arbitrary cross section including the adhered state of the culture substrate 2 and the cells 3 can be obtained with almost no state. FIG. 5 shows images observed by changing the angle of the culture substrate 2.

Although water 4 is used for culturing cells 3 in the present embodiment, it is not limited to this form, and a commonly used culture solution may be used. The ingredients and concentrations may be adjusted according to. Furthermore, the conditions and methods of the hydrophilic treatment and the pretreatment of culture may vary depending on the specimen to be observed and the content of the experiment, and in that case, they may be appropriately changed and used.

FIG. 6 is a diagram showing an example of a mechanism for changing the angle of the culture substrate 2 with respect to the optical axis.

As shown in (1) of FIG. 6, in this mechanism,
A spherical movable member 9 having a concave portion is fitted to a base 8 mounted on the microscope stage, and the movable member 9 is configured to be spherically rotatable by a spherical sliding portion 10.
Then, the culture substrate 2 on which the cells 3 are placed is held in the concave portion of the movable member 9, and the concave portion is filled with water 4.

When adjusting the inclination of the culture substrate 2, FIG.
As shown in (2), the movable member 9 is rotated to change the angle of the cell 3 with respect to the optical axis. In order to reduce the influence of the rotation on the observation, it is desirable that the cell 3 is placed at a position substantially coincident with the rotation center of the movable member 9.

Here, the rotation of the movable member 9 may be performed manually by a microscopist or may be operated by a driving device (not shown). Further, the movable member 9 need not be a spherical shape but may be configured by using a cylindrical shape having a circular cross section.

FIG. 7 is a diagram showing a second embodiment of the observation method according to the present invention. In this figure, parts having the same functions as in FIG. 2 are assigned the same reference numerals and detailed explanations thereof are omitted.
In the present embodiment, in order to reduce the influence of gravity acting on the cells 3 at the time of observation, the optical axis of the water immersion objective lens 1 is arranged horizontally unlike the first embodiment.

The tip of the water immersion objective lens 1 is inserted into the container 11 and is sealed with an O-ring 12 so that the water immersion objective lens 1 can be moved horizontally to adjust the focus. ing. The inside of the container 11 is filled with the culture solution 41, and further a pipe (not shown) for introducing carbon dioxide gas for culturing the cells 3 is connected. Also, container 1
1 is a culture substrate 2 loaded with cultured cells 3
It is rotatably attached via 3a and 13b,
The tilt can be changed by an external operation.

In the configuration of this embodiment, since the culture substrate 2 can be always kept in the vicinity of the horizontal, cells can be cultured and observed in a more natural manner, and like the first embodiment, It is possible to obtain an image of an arbitrary cross section with almost no deterioration in imaging performance.

FIG. 8 is a diagram showing a third embodiment of the observation method according to the present invention. In this figure, parts having the same functions as in FIG. 2 are assigned the same reference numerals and detailed explanations thereof are omitted.

This embodiment differs from the first embodiment in that
Both sides of the culture substrate 2 are subjected to the above-mentioned hydrophilic treatment, and cells 3a and 3b are cultured on both sides thereof. The needle 7 is a hollow pipe, one end of which is connected to a syringe for injecting a drug, and the other end of which is arranged in the vicinity of the cell 3b and used for injecting a drug for stimulating cells.

With this configuration, as shown in FIG. 9, cells in two environments separated by a substrate can be observed at the same time, and at the same time, one of the cells can be observed with a reagent or the like using a needle 7. By stimulating only the cells, it becomes possible to compare and observe the state of cells with and without stimulation.

FIG. 10 is a diagram showing a fourth embodiment of the observation method according to the present invention. In this figure, parts having the same functions as in FIG. 2 are assigned the same reference numerals and detailed explanations thereof are omitted.

In the present embodiment, unlike the first embodiment, the culture substrate 23 having a substantially hemispherical shape is rotatably attached to the bottom opening of the container 21 via an O-ring 24. The flat surface side of the culture substrate 23 is subjected to a hydrophilization treatment and a culture treatment to culture the cells 3. Further, a handle 22 for tilting operation is attached to the spherical surface side of the culture substrate 23, and further, an illumination device 25 is provided below the culture substrate 23.
And an optical system for illumination (not shown).

By thus forming the culture substrate 23 in a hemispherical shape and constituting a part of the illumination optical system, even if the handle 22 for tilting is operated, the angle of the cell 3 can be changed. There is no change in the illumination state from the lower surface of the cell, and a bright observation image can always be obtained.

As described above, the aforementioned problems can be achieved by using the respective embodiments, but the respective embodiments described above are particularly effective for observing cultured cells. That is, the polar structure of the cultured cells develops perpendicular to the culture substrate. Conventionally, information in the Z-axis direction was obtained by moving the stage of a scanning laser microscope, but this conventional method had a limit in observation. However, by using each of the embodiments, information on cell polarity can be observed with high accuracy and speed.

[0045]

As described above, according to the present invention, when observing a biological specimen using a scanning laser microscope equipped with a water immersion objective lens, particularly a high magnification / high numerical aperture water immersion objective lens. In addition, it is possible to obtain a culture substrate for microscopic observation in which a biological specimen can be cultured and the imaging performance is less deteriorated, and an observation method suitable for a biological specimen can be obtained using the culture substrate.

[Brief description of drawings]

FIG. 1 is a schematic flowchart illustrating a method for modifying a culture substrate for microscopic observation according to the present invention.

FIG. 2 is a diagram showing an embodiment of an observation method according to the present invention.

FIG. 3 is a view showing an image observed by a microscope.

FIG. 4 is a diagram showing another embodiment of the observation method according to the present invention.

FIG. 5 is a diagram showing an image observed by a microscope.

FIG. 6 is a view showing a mechanism for changing the angle of the culture substrate with respect to the optical axis.

FIG. 7 is a diagram showing another embodiment of the observation method according to the present invention.

FIG. 8 is a diagram showing another embodiment of the observation method according to the present invention.

FIG. 9 is a diagram showing an image observed by a microscope.

FIG. 10 is a view showing another embodiment of the observation method according to the present invention.

[Explanation of symbols]

1. Water immersion objective lens 2 ... Culture substrate 3 ... cells 3a ... cell 3b ... cell 4 ... water 7 ... Needle 8 ... Base 9 ... Movable member 12 ... O-ring 22 ... Handle 23 ... Culture substrate 24 ... O-ring 25 ... Lighting device 41 ... Culture solution

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shuzo Mishima             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. (72) Inventor Noriyuki Shimizu             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. (72) Inventor Hiroaki Kasai             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. F-term (reference) 2G045 AA24 CB01 FA16 JA07                 2H052 AA08 AA09 AB02 AC15 AC34                       AD22 AE03 AE13

Claims (4)

[Claims] 1. A culture substrate for microscopic observation used for observing a biological specimen using a water immersion objective lens, which is made of a transparent fluororesin having a refractive index of 1.33 or more and 1.34 or less, and the substrate surface is hydrophilized. A culture substrate for microscopic observation, which is characterized in that 2. The culture substrate for microscopic observation according to claim 1, wherein an ultraviolet light source is used for the hydrophilic treatment of the substrate surface. 3. The culture substrate for microscopic observation according to claim 1, wherein a part of the surface of the substrate is hydrophilized. 4. The method for observing a biological specimen using the culture substrate for microscope observation according to claim 1, wherein the culture substrate for microscope observation is arranged at an arbitrary angle with respect to the optical axis of the water immersion objective lens. A method for observing a biological specimen, characterized by observing.