JPH03223710A - Microscope - Google Patents

Abstract

PURPOSE:To put an inspection system which uses a porous filter to practical use by arranging a light diffusing layer between a specimen and a lighting lens. CONSTITUTION:The light diffusing layer is arranged between the specimen and lighting lens. Namely, the light diffusing layer 6 is positioned between the specimen system 5 where the porous filter 3 catching the specimen is arranged on transparent slide glass as it is and a microscopic lighting system including a condenser lens 10 for lighting. When the specimen 2 caught by the porous filter 3 is observed through the microscope, the light from a light source is scattered to make the contours of the holes of the porous filter 3 invisible. Consequently, the microscopic inspection system which uses the porous filter can be obtained as a really practical system.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a microscope or a microscope component used for biological and medical microscopic observation of living cells, bacteria, viruses, etc., for example.

[Conventional technology]

In general, microscopic observations for biological and medical research such as cell examinations, confirmation of bacteria, and drug efficacy tests are carried out by placing the specimen on a glass slide so that it does not overlap, and placing the specimen on a slide that is likely to fall off. This is done through a highly skilled, laborious, and time-consuming process in which the specimen is fixed on glass, stained, washed, and then subjected to microscopic observation.

Due to the actual state of the above processing, recently, a test in which a porous filter is used to capture the specimen in the culture medium, and the specimen is fixed on the porous filter, stained, and washed is subjected to microscopic observation. The system is attracting attention. According to this new testing system, it is possible to easily capture specimens without overlapping them, and the specimens on the porous filter are difficult to fall off, making fixation, staining, and washing processes easier. Not only does it not require the same level of skill as processing, it is also expected to significantly reduce labor and processing time.

By the way, if the above-mentioned porous filter that captures the specimen is placed on a transparent slide glass and observed under a microscope, the outline of the pores of the porous filter will overlap with the specimen, which will interfere with the observation.

To prevent this, it is possible to transfer the sample captured on a porous filter onto a transparent slide glass before observation, or to remove the porous filter by dissolving it with a solvent on a transparent slide glass before observing it. This is being done in some areas. However, these methods require skill and a great deal of effort and time to transfer and remove the solution without causing the specimen to fall off.
It is not practical.

Therefore, in order to prevent observation problems caused by the pores of the porous filter without requiring such skill, effort, and time, the porous filter that has captured the sample can be placed on a translucent slide glass and observed as it is. It has been proposed to do so (Japanese Unexamined Patent Publication No. 64-6914).

The above-mentioned observation is characterized by the fact that a translucent slide glass that can scatter light is used instead of a transparent slide glass used in boat fishing. The purpose of using such a translucent slide glass is to scatter the light from the light source of the microscope, making the ring of the porous filter optically invisible.

[Problem to be solved by the invention]

However, the above-mentioned translucent slide glass is more expensive than conventional slide glass, and costs several to several tens of times more. Moreover, one translucent glass slide must be used for each subject, which is therefore uneconomical.

Furthermore, the above-mentioned Japanese Patent Application Laid-Open No. 64-6914 discloses that the outline of the pores of a porous filter is made invisible during observation by using a translucent slide glass.
Although the basic principle is disclosed, it is not specifically disclosed whether such a translucent slide glass is effective.

Translucent slide glass does not simply mean that the conventionally widely used transparent slide glass has been made translucent, but rather it has a light-diffusing effect that makes it difficult to see the outline of the pores of a porous filter. At the same time, it is necessary to satisfy the requirement of securing transmitted light that provides sufficient brightness of the field of view even under high magnification.

As mentioned above, although inspection systems using porous filters have the potential to fundamentally improve the inefficient work that has been done up until now, they are uneconomical and Currently, it is not a truly practical inspection system because a translucent slide glass is not provided.

The present invention has been made in view of the current situation, and an object of the present invention is to provide a microscopic inspection system that has the same effect as when using the slide glass described above and satisfies the above requirements.

[Means and effects for solving the problems] That is, the present invention is a microscope in which a light diffusion layer 6 is disposed between the subject 2 and the illumination lens 10.

The present invention will be explained in detail based on FIGS. 1 to 3.

For example, as shown in FIG. 1, the light diffusing layer 6 of the present invention is installed directly under the subject system 5 in which the porous filter 3 that captures the subject 2 is placed on a transparent slide glass 4. , and a microscope illumination light system including a condenser lens 10 for illumination, etc., and scatters light from the light source when observing the subject 2 captured by the porous filter 3 under a microscope. filter 3
The purpose is to make the outline of the hole invisible.

1 is a cover glass, 8 is a sample stage, and 9 is a frame.

The distance between the light diffusing layer 6 and the porous filter 3 of the present invention is preferably as short as possible, preferably 0 or 2 cm or less, preferably 1 day or less, and more preferably 0.5 aun or less. Furthermore, there are cases where the slide glass 4 is not required. If the light diffusion layer 6 is located more than two feet away from the porous filter 3, the contours of the pores will appear to overlap with the subject 2. The surface of the light diffusing layer 6 relative to the subject system 5 may be a plane parallel to the system 5.

The semi-transparent portion 6 of the present invention preferably has a total light transmittance of 30% or more, a parallel light transmittance of 6% or less, and a haze degree of 85% or more, although this varies depending on the selection and combination of materials used. Currently available materials have a total light transmittance of 30 to 60.
%, more preferably 45 to 55%. Further, the parallel light transmittance is 1 to 4%, preferably 2 to 3.
3% or more preferred. More preferably, the degree of haze is 90% or more. These conditions are intended to obtain light scattering necessary to make the contours of the pores of the porous filter 3 invisible and to obtain a brightness of view that facilitates observation at high magnification. Outside these ranges, the outline of the hole tends to overlap with the subject, making it easier to see, or the field of vision tends to become darker.

Particularly important among these characteristics is the degree of haze. If the haze level is the same but the total light transmittance is large and the parallel light transmittance is large, adjust the irradiation light dose and reduce it, so that the outline of the hole overlaps with the object and becomes easier to see. , your vision will not become dark. Also,
In order to make the outline of the pores difficult to see, there has been an attempt to pour a liquid with a refractive index equivalent to that of Filter 3 into the pores, and although it is insufficient, it seems to be effective, and if this method is adopted, the haze will be reduced. It is possible to use a light-diffusing layer 6 with a lower intensity, or to make the field of view brighter so that the contours of the holes are not visible.

The light-diffusing layer is made of a solid silicone resin in which a silicon atom is directly connected to an organic group having an affinity for the coating film-forming material or its monomer, forming a polysiloxane bond, as disclosed in, for example, Japanese Unexamined Patent Publication No. 1-172801. It is a resin obtained by curing a photocurable resin containing silicone resin spherical particles having a number average particle diameter of 0.3 to 10 μm. Either only the light diffusion layer 6 itself is disposed, or it is placed on the transparent substrate 7.
The latter is preferred. The light diffusing layer as described above has excellent surface hardness, surface smoothness, and chemical resistance.

The surface of the light diffusing layer preferably has a surface roughness of 1 μm or less so that the focus does not shift even if the observation position is shifted during microscopic observation.

As the photocurable resin, various conventionally known ones can be used, and there are no particular limitations, and it is preferable to use an ultraviolet curing type.

Examples of the ultraviolet curable photocurable resin include those whose main components are a prepolymer, a monomer, a photopolymerization initiator, and a solvent.

Examples of the above-mentioned prepolymers include low-molecular oligomers in which acryloyl groups are introduced at the ends or chains of molecules such as polyurethane acrylate, polyester acrylate, polyepoxy acrylate, polyether acrylate, and polyol acrylate.

Examples of monomers include PETA (pentaerythritol tetraacrylate), TMPTA (trimethylolpropane triacrylate), and 1.6-HDDA.
(1°6 hexanedimooldiacrylate), NPG
DA (neopentyl glycol diacrylate), 2E
Examples include HA (2-ethylenehexyl acrylate).

Examples of photopolymerization initiators include benzoin-based compounds such as benzoin methyl ether and benzoin butyl ether;
．． Ketones such as 4-jetoxyacetophenone, p-chlorobenzophenone, benzophenone, azobenzenes such as abbisisobutylnitrile, 2,2'-azobis-(2'4-dimethylvaleronitrile) azobenzene, quinones such as anthoraquinone, phenatochiranone, etc. Photopolymerization initiators such as

Examples of solvents include aromatic solvents such as troene and xylene, alcohol solvents such as isopropanol, ethyl acetate,
Examples include ester-based solvents such as butyl acetate, and ketone-based solvents such as MEK.

The number average particle diameter of the silicone resin spherical particles is 0.3.
It is preferably 4.0 μm to 4.0 μm, and particularly preferably around 2 μm.

By combining silicone spherical particles in this range and photocurable resin, it is easy to obtain a light diffusing layer 7 with high surface hardness, and it is easy to balance total light transmittance, parallel light transmittance, and haze degree. In addition, the smaller the number average particle diameter of silicone resin spherical particles, the smaller the total light transmittance, and the larger the number, the higher the total light transmittance.The total light transmittance, parallel light transmittance, and haze degree are It can also be freely controlled by selecting the amount of silicone resin spherical particles, which will be described later.

The number average particle diameter of the silicone resin spherical particles is determined by the following measurement method.

Measuring device: Centrifugal automatic particle size distribution measuring device (particle analyzer) Format: CAPA-500 Measuring method: Calculated by light transmission liquid phase sedimentation particle size distribution measuring method that employs high-speed centrifugal sedimentation method and natural sedimentation method Dispersion medium: Interface Activator aqueous solution dispersion conditions: The amount of ultrasonically dispersed silicone resin spherical particles added to the photocurable resin is as follows:
50 to 15 parts by weight per 100 parts by weight of solid content of photocurable resin
It is preferable that the amount is 0 parts by weight.

If the amount added is less than 50 parts by weight, the thickness of the light diffusing layer in the solvent-containing state will be 2.
80 μm or more (150 μm or more after curing) is required,
This makes it difficult to form a light diffusion layer.

On the other hand, if the amount added is too large, it becomes difficult to form a light diffusing layer.

The transparent substrate 7 on which the light diffusion layer 6 is disposed may be a soda glass plate similar to that used as a general slide glass, or other transparent plate materials such as a synthetic resin plate such as polymethyl methacrylate. But that's fine. In particular, a borosilicate glass plate is preferable because it has good ultraviolet transmittance and is effective for fluorescence microscopic observation.

As a method for forming a light diffusing layer using a photocurable resin containing silicone resin spherical particles, it is preferable to use a transfer method because surface smoothness is easily obtained.

This transfer method will be explained below with reference to FIG.

First, as shown in FIG. 3(a), the support film 1
1 is coated with the above-described photocurable resin to which silicone resin spherical particles are added. This coating can be done by any method as long as the photocurable resin containing spherical silicone resin particles can be uniformly deposited on the support film 11, but it is easy to do this by using a normal coating method using a reverse coater, comma coater, air knife coater, etc. It can be done.

As the support film 11, a film is used that has a smooth surface and can be peeled off after the photocurable resin is cured, and usually has a thickness of 50 to 180 μm. A specific example is a polyethylene terephthalate film.

After completing the coating on the support film 1, the photocurable resin is sufficiently dried and the solvent contained therein is removed. If necessary, the cover film I2 is covered and laminated as shown in FIG. 3(b). The applied photocurable resin containing spherical silicone resin particles may be protected.

The cover film 12 is a film that can be easily peeled off from the applied photocurable resin containing spherical silicone resin particles and has a smooth surface, such as a polyethylene film.

Using the transfer sheet obtained above (the cover film is peeled off), the photocurable resin containing spherical silicone resin particles is transferred onto the transparent substrate 7, as shown in FIG. 3(C).

This transfer can be performed by thermal transfer by subjecting the surface of the transparent substrate 7 to an anchor treatment if necessary. Thermal transfer can be performed by pressing and bonding with a roller 13 heated to 60 to 140°C. In addition, hot stamping can be used industrially.

After the above transfer, as shown in FIG. 3(d), the photocurable resin is photocured, and the support film is peeled off to form a translucent part (slide glass stand) 1 having a light diffusion layer 6.
You can get 4.

Various materials other than these can be used as the light diffusion layer 6, but as an example other than the above, a translucent crystalline resin can be used.

Various conventionally known translucent crystalline resins can be used for the light diffusion layer 6, and there are no particular restrictions, but resins with a high degree of crystallinity are preferred, and engineering plastics such as polyacetal, polyester, and polyamide are preferred. Alternatively, a general-purpose plastic such as high-density polyethylene is preferable.

More preferred are polyacetals which are resins with a particularly high degree of crystallinity, and resins highly crystallized or microcrystalized with a nucleating agent, particularly polyacetal resins microcrystallized to have an average crystal particle size of 0.3 to 50 μm. etc. are preferred.

The finer the average particle size of the crystals of this crystalline resin, the more uniformly diffused light can be obtained. The total light transmittance, parallel light transmittance, and haze degree can be controlled by the crystallinity degree and thickness as well as the average particle diameter, and the higher the crystallinity degree, the thinner the thickness can be.

As for the manufacturing method of this semi-transparent part 14, extrusion molding method, injection molding method, etc. which are general manufacturing methods of resin can be used.

The translucent part 14 is attached to the microscope by being fixed or placed in a conventional manner on the sample stage 8 on which the object system 5 is placed, or by being fixed on the top of the condenser lens 10 for illuminating light. The translucent part 14 may be placed on the sample stage 8 or the condenser lens 10 and moved with the object system 5 when determining the observation location. It is also possible to be rejected.

〔Example〕

Example 1 A translucent slide glass stand 14 having a light diffusing layer 6 was produced using the following materials and method.

(1) Transparent substrate 7 Transparent plate D Niacrylic plate (900 μm thick) Transparent plate E Niborosilicate glass plate (885 μm thick) Transparent plate F Niborosilicate glass plate (1010 μm thick) Both sizes are 10
It was set to 0x100 mm.

(2) The light-diffusing layer photocurable resin includes polyurethane acrylate and T.
An ultraviolet curable 50 wt % photocurable resin solution containing MPTA, p-chlorobenzophenone, and a solvent prepared by mixing toluene and butyl acetate in a ratio of 1:1 was used.

The number average particle diameter of the silicone resin spherical particles is 2 μm.
I used the one from

Table 1 shows the blending ratio of the photocurable resin and silicone resin spherical particles. In addition, the unit is weight %, and the photocurable resin is the weight % of solid content.

Table 1 (3) Preparation method Using a polyethylene terephthalate film with a thickness of 125 μm as a support film, apply the light diffusion layer material of the above composition to a thickness of 230 μm using a bar coder, and then
It was dried in an air bath for 3 minutes under a C atmosphere, and a 60 μm thick polyethylene film was laminated thereon as a cover film.

After peeling off the polyethylene film of the transfer sheet obtained above from four anchor-treated transparent substrates 7,
This peeled surface is stamped using a hot stamping machine (roll temperature 100°C).
), and was thermocompression bonded to the arranged transparent substrates 7.

Next, after photocuring the photocurable resin by irradiating ultraviolet rays from the polyethylene terephthalate film side, the polyethylene terephthalate film is peeled off, and the light diffusion layer 6 is
And the translucent part (slide glass stand) 1 where this is installed
I got 4.

The total light transmittance, parallel light transmittance, haze degree, and surface roughness of the light diffusion layer 6 of the semi-transparent part 14 obtained above were measured using the following apparatus and method.

Optical property measurement: Digital haze meter TC-HI manufactured by Tokyo Denshoku (model (based on JIS K-6714, 6718°6719 and ASTM-D-1003) Surface roughness measurement: Stylus type surface manufactured by Tokyo Seimitsu Co., Ltd. Roughness meter rSurfco
554AJ (based on JIS-B-0601) The results of the above measurements are shown in Table 2.

Table 2 On the other hand, a porous filter 3 with a frame was prepared using the materials and method described below.

(1) Frame 9 A rectangular stainless steel plate with a thickness of 150 μm and a diameter of 21 mm was punched in the center of a 25×29 mm square stainless steel plate with rounded corners.

(2) Porous Filter 3 A polycarbonate film with a thickness of 7 μm and straight pores of 3 μm in diameter was used (Nuclipore Filter manufactured by General Electric Company).

(3) Creation method A hot melt film is pressed and heated on the frame 9 (at a temperature of 100 to 160°C), the porous filter 3 is placed on top of it, and the film is suppressed and heated again (at a temperature of 100 to 160°C) By doing so, the two were joined to create a porous filter with a frame 3.

Using the frame-attached porous filter 3 obtained above and the slide glass 4, a cell specimen was prepared in the following manner.

First, biological tissue was cut into thin sections with a razor, placed in culture medium, sucked into a syringe with a needle attached, and single cells were obtained by rapidly extruding the tissue into the culture medium.

Next, the prepared porous filter with frame 3 was placed on the filter, and the single cells in the culture solution obtained above were filtered. The cells captured by the frame-attached porous filter 3 are fixed, stained, and washed with the porous filter 3, and then the cells are reduced to a thickness of 3 mm, 1.2 mm, and 0.7 mm.
It was placed on each slide glass 4 of 0.4mm, 0.4mm, and 0.15mm, and observed at 50x, 100x, 200x, and 1000x magnification using a microscope equipped with the slide glass stand 14. went.

As a result, it was confirmed that cell specimen preparation was extremely easy and workability was excellent. Also, the thickness is 0.15mm,
When slide glasses 4 of 0.4 mm, 0.7 mm, and 1.2 mm are used, light diffusion layers A, B, and C are provided, and E.

In any combination with the transparent substrate F, the pores of the porous filter 3 were not visible and could be clearly observed at any magnification. Although the pores of the porous filter 3 were slightly observed in the slide glass 4 having a thickness of 3 mm, the cell specimen could also be clearly observed.

Furthermore, when observing without using the slide glass stand 14, the pores of the porous filter appeared to overlap with the cells, making detailed observation difficult.

Example 2 Light diffusing layer 6 made of crystalline resin using the following materials and method
A translucent slide glass stand 14 having the following was created.

(1) Transparent substrate 7: See Example 1 (2) Light diffusion layer Light diffusion layer G: Sheet made of microcrystalline polyacetal (manufactured by Asahi Kasei, crystallinity 70.1%, crystal grain size 2-3 μm) (850 μm thick) , extrusion molded product) Light diffusion layer H: Boriacetal (Tenac 30tO.

Made by Asahi Kasei, injection molding grade, crystallinity 69.0%, crystal grain size 30-40 μm) sheet (830 μm thick, extrusion molded product) Light diffusion layer ■: Nylon 6
6 (Leona 1300. Manufactured by Asahi Kasei, injection molding grade, crystallinity 34.8%, crystal grain size 30-40 μm) sheet (1
820 μm thick, injection molded product) Light diffusion layer J: Microcrystalline polyethylene (Asahi Kasei; light diffusion layer: Light diffusion layer L: Light diffusion layer M: Light diffusion layer N: Crystallinity 70.2%, crystal grain size 40-50 μm) (1320 μm thick, injection molded product) Polyethylene (Santic f (DJ240, manufactured by Asahi Kasei,
Injection molding grade, crystallinity 69.6%, crystal grain size 100-200 μm) sheet (2280 μm, injection molded product) Polyethylene (Santic HDJ240, manufactured by Asahi Kasei, injection molding grade, crystallinity 69.6%, crystal grain Sheet made of microcrystalline polyacetal (manufactured by Asahi Kasei, crystallinity 69.7%, crystal grain size 2-3 μm) (580 μm, injection molded product) Polyacetal (Nanatsuku 5o1O1) Sheet (620 μm, injection molded product) manufactured by Asahi Kasei, crystallinity 69.7%, crystal grain size 30 to 50 μm) Each size was 100 mm x 100 mm.

(3) Creation method Ten types of translucent parts (slide glass stands) 14 were created by adhering and combining the three transparent substrates 7 and the light diffusion layer 6 with double-sided tape (manufactured by Chiban Co., Ltd.).

The total light transmittance, parallel light transmittance, and haze degree of the semitransparent portion 14 obtained above were measured using the apparatus and method of Example 1.

The results of the above measurements are shown in Table 3.

On the other hand, a porous filter with a frame 3 was produced by the method of Example 1.

A cell specimen was prepared by the method of Example 1 using the frame-attached porous filter 3 obtained above and the slide glass 4.

These cell specimens were magnified at 50x, 100x, 200x, and 1x using a microscope equipped with the slide glass table 14.
Each observation was made under 1,000 times magnification.

As a result, it was confirmed that cell specimen preparation was extremely easy and workability was excellent. In addition, Example (light diffusion layer F-K
The pores of the porous filter 3 were not visible on any of the slide glass stands 14 (SN), and could be clearly observed at any magnification.

When using the light diffusion layer M, the pores of the porous filter were visible and detailed observation was difficult, but by suppressing the light amount (voltage) to about 80% (10V → 8V) The pores of the cell filter 3 were visible, and although it took some time to observe the cell specimen in detail, it could be observed at any magnification.

Furthermore, when a slide glass stand was not used, the pores of the porous filter 3 appeared to overlap with the cells during observation, making detailed observation impossible.

〔Effect of the invention〕

As explained above, the present invention makes it possible to make a microscopic examination system using a porous filter into a truly practical system by using a light diffusion layer, and through the spread of this system, the microscopic examination can be performed quickly and reliably. This makes it possible to

[Brief explanation of drawings]

1st. Figure 2 is a partial cross-sectional view of a microscope having a light diffusing plate 6 according to the present invention, including a specimen system 5 equipped with a slide glass table 15. This is the case where the entire transparent part is a semi-transparent light-diffusing layer. FIGS. 3(a) to 3(d) show a semi-transparent part 14 formed by a transfer method.
FIG. ■=Cover glass, 2: Subject, 3: Porous filter 4: Transparent slide glass, 5: Subject system, 6:
Light diffusion layer, 7: Transparent substrate, 8: Sample stage, 9: Frame, lO
: Illumination lens (condenser lens), 11: Support film, 12: Cover film, 13: Roller 14:
Translucent part (slide glass stand).

Claims (1)

[Claims] 1 A microscope in which a light diffusion layer is provided between the subject and the illumination lens.