JPH10123429A - Slide glass and cover glass - Google Patents

Abstract

PROBLEM TO BE SOLVED: To observe a sample by having excellent signal light intensity(S)/ background noise light(B) by providing a reflection preventive film at a whole or a part of one surface of a transparent plate and a discriminating mark for discriminating one surface. SOLUTION: The reflection preventive film 12 is formed on one surface of the transparent plate 11 at a slide glass 10, and the discriminating mark 13 for discriminating a surface where the reflection preventive film 12 is formed is marked. The reflection preventive film 12 is formed on one surface of the transparent plate 11, and a refractive index and a film thickness are appropriately designed in accordance with the respective refractive indexes of the transparent plate 11 and an air layer so as to make a reflectance small for the light of a specified wavelength. In this case, the specified wavelength is excitation light wavelength in the case of being used for fluorescent observation though it depends on the use of the slide glass 10. Also, the surface where the discriminating mark 13 is put may be the surface of the transparent plate 11 where the reflection preventive film 12 is formed, the surface on an opposite side or on the reflection preventive film 12.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a slide glass and a cover glass suitable for use with an optical microscope, particularly a fluorescence microscope.

[0002]

2. Description of the Related Art Conventionally, a sample labeled with a fluorescent probe has been observed with a fluorescence microscope. At this time, the sample is sandwiched between a slide glass and a cover glass, the sample is placed on a sample stage of a fluorescence microscope, and the sample is irradiated with excitation light to detect fluorescence generated in the sample. Observe the sample.

[0003]

In such a fluorescence microscope, not only fluorescence is generated in the sample, but also autofluorescence is generated in the optical system of the fluorescence microscope. This auto-fluorescence becomes a noise when observing the fluorescence from the sample. Therefore, in order to observe the fluorescence generated from the sample with a good S / B ratio, it is necessary to suppress the auto-fluorescence as small as possible. is there. Here, the S / B ratio is an intensity ratio between the signal light intensity (S) and the background noise light (B), and is used as a measure of the performance related to the measurement sensitivity of the optical system.

The auto-fluorescence is generated by various factors in the optical system of the fluorescence microscope. The auto-fluorescence is generated not only when the excitation light passes through the objective lens but also due to a slide glass or a cover glass on which the sample is placed. It is thought that it will occur even if it occurs. Therefore, in order to suppress the auto-fluorescence, it is necessary to suppress the generation of auto-fluorescence caused by the slide glass and the cover glass on which the sample is placed.

[0005] Such a problem is not limited to the fluorescence microscope but also exists in other optical microscopes. That is, the illumination light applied to the sample from the objective lens is reflected and scattered by something other than the sample, and the reflected light
When the scattered light enters the objective lens again, the S / B ratio for sample observation is degraded.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has as its object to provide a slide glass and a cover glass capable of observing a sample with an excellent S / B ratio.

[0007]

The slide glass according to the present invention has an antireflection film on all or a part of one surface of a transparent flat plate, and has an identification mark for identifying one surface. It is characterized by. According to this slide glass, since the reflectance on the surface of the transparent flat plate on which the antireflection film is formed is reduced, the S / B ratio in sample observation is improved, and the surface of the transparent flat plate on which the antireflection film is formed is also provided. Identification is facilitated by the identification mark.

If the antireflection film of the slide glass reduces the reflectance with respect to the excitation light, and if the transparent flat plate is a quartz glass plate, it is suitable for use with a fluorescence microscope.

When the anti-reflection film of the slide glass is designed to reduce the reflectance with respect to substantially perpendicularly incident light, it is suitable for use with a bright field illumination microscope. The incident angle is 53 ° or more 8
When designed to reduce the reflectance with respect to light incident in the range of 2 ° or less, it is suitable for use with a dark-field illumination microscope.

[0010] The cover glass according to the present invention is characterized in that the transparent glass has an antireflection film on the whole or a part of one surface thereof, and has an identification mark for identifying the one surface. According to this cover glass, the reflectance on the surface of the transparent flat plate on which the antireflection film is formed is reduced, so that the S / B ratio in sample observation is improved, and the surface of the transparent flat plate on which the antireflection film is formed is also provided. Identification is facilitated by the identification mark.

If the antireflection film of the cover glass reduces the reflectance with respect to the excitation light, and if the transparent flat plate is a quartz glass plate, it is suitable for use with a fluorescence microscope.

When the anti-reflection film of the cover glass is designed to reduce the reflectance with respect to substantially perpendicularly incident light, it is suitable for use with a bright-field illumination microscope. The prevention film has an incident angle of 53 ° or more 82
場合 When designed to reduce the reflectance for light incident in the following ranges, it is suitable for use with a dark-field illumination microscope.

[0013]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

First, prior to the description of the slide glass and cover glass according to the present invention, the results of analyzing the causes of auto-fluorescence in a fluorescence microscope will be described. In a fluorescence microscope, it is said that auto-fluorescence generated in an objective lens most affects the S / B ratio. In this objective lens, not only does the autofluorescence occur when the excitation light passes through the objective lens from the excitation light source toward the sample, but also the excitation light once enters the objective lens from the sample side after passing through the objective lens. It is considered that the auto-fluorescence is also generated by doing so.

FIG. 6 is an explanatory diagram of excitation light reflection in a fluorescence microscope. Excitation light A emitted from an excitation light source (not shown) passes through the objective lens 4, passes through the cover glass 2, and irradiates the sample 3. Then, the fluorescence generated in the sample 3 passes through the cover glass 2 again, passes through the objective lens 4, and is detected by a detector (not shown). In addition,
An antireflection film for fluorescence 4a is provided on the sample 3 side of the objective lens 4 in order to suppress the reflection of the fluorescent light and make the incident light efficiently.

However, the excitation light A not only irradiates the sample 3 but also reflects on each boundary surface, and the reflected light is incident on the objective lens 4 again. For example, reflected light B1 is generated at a boundary surface between the objective lens 4 and the antireflection film for fluorescence 4a, and reflected light B2 is generated at a boundary surface between the cover glass 2 and an air layer thereon. Reflected light B3 is generated at the boundary surface with the lower air layer, and reflected light B4 is generated at the boundary surface between the condenser lens 5 and the upper air layer. In addition, reflected light of the excitation light A is generated at the boundary between the sample 3 and the cover glass 2 and also at the boundary between the sample 3 and the slide glass 1. In this figure, for convenience of explanation, the incident excitation light A is represented by a straight line, and each of the reflected lights B1 to B4 is represented obliquely.

When these reflected lights enter the objective lens 4 again, autofluorescence is generated in the objective lens 4 due to this. According to the experiment by the inventor, it was confirmed that when the reflectance of the excitation light A at the interface between the objective lens 4 and the antireflection film for fluorescence 4a increases by 4 to 5%, the autofluorescence increases by about 10%. . In addition, the amount of autofluorescence generated due to the reflection of the excitation light A decreases as the reflection surface of the excitation light A moves away from the objective lens 4, but the excitation light at the boundary surface between the slide glass 1 and the air layer thereunder. It was confirmed that the auto-fluorescence increased by several percent even by the reflection of A.

The present invention has been made based on the results of the above-described analysis, and is based on the reflection of the excitation light at each of the interface between the slide glass and the air layer and the interface between the cover glass and the air layer. The purpose of the present invention is to reduce the generation of autofluorescence in an objective lens.

Next, the slide glass according to the present invention will be described. FIG. 1 is a perspective view of a slide glass according to the present invention.

The slide glass 10 according to the present invention has an anti-reflection film 12 formed on one surface of a transparent flat plate 11, and an identification for identifying the surface on which the anti-reflection film 12 is formed. The sign 13 is marked. Generally, a glass plate is used for the transparent flat plate 11. However, in consideration of a case where a sample is placed on the slide glass 10 and the sample is irradiated with excitation light to observe fluorescence generated, a non-fluorescent plate is used. A quartz glass plate, which is a conductive material, is preferable.

The anti-reflection film 12 is formed on one surface of the transparent flat plate 11, and has a refractive index and a film thickness of each of the transparent flat plate 11 and the air layer such that the reflectance is small with respect to light of a predetermined wavelength. Appropriately designed according to the refractive index. Here, the predetermined wavelength is an excitation light wavelength when used for fluorescence observation, although it depends on the use of the slide glass 10.
The anti-reflection film 12 may be formed on one entire surface of the transparent flat plate 11 as shown in this figure, or may be formed partially, for example, in the center of the surface on which the sample is placed. It may be formed only in the region.

As described above, the antireflection film 12 is formed on the transparent flat plate 11.
However, it is not easy to identify the surface of the transparent flat plate 11 on which the antireflection film 12 is formed. Therefore, an identification mark 13 is provided to facilitate the identification. The surface on which the identification mark 13 is written is the transparent flat plate 1.
It may be a surface on which one antireflection film 12 is formed,
The surface on the opposite side may be used.
2 may be on. The position where the identification mark 13 is written is
The periphery of the transparent flat plate 11 is preferable so as not to hinder the sample observation. Further, the identification marker 13 may be a character, a graphic, a symbol, a trademark, a logo of a manufacturer name, or any other marker, and may be displayed not only by printing but also by engraving.

Since the slide glass has a thickness of about 1 mm, it can be determined which side of the transparent flat plate 11 has the identification mark 13 on it. It is preferable to adopt an identification mark 13 having a shape different from each other when viewed from the other side.

Next, the usage of the slide glass according to the present invention will be described. FIG. 2 is a diagram illustrating the use of the slide glass according to the present invention in a fluorescence microscope.

In this figure, a sample 30 is sandwiched between a surface of the slide glass 10 according to the present invention on which the antireflection film 12 is not formed and the conventional cover glass 20A, and this is placed on a sample stage (not shown). And irradiates the sample 30 with the excitation light A using the objective lens 40 and observes the fluorescence generated in the sample 30.

In this case, when the sample 30 is irradiated with the excitation light A, the reflected light B1
Is generated, and reflected light B2 is generated at the interface between the conventional cover glass 20A and the air layer, and reflected light B4 is generated at the interface between the condenser lens 50 and the air layer. However, reflected light is reduced at the interface between the slide glass 10 and the air layer according to the present invention. Due to this reduction in reflected light, auto-fluorescence is several percent.
It was confirmed that it decreased.

Next, the cover glass according to the present invention will be described. FIG. 3 is a perspective view of the cover glass according to the present invention.

The cover glass 20 according to the present invention has an anti-reflection film 22 formed on one surface of a transparent flat plate 21 and further has an identification for identifying the surface on which the anti-reflection film 22 is formed. The sign 23 is marked. Generally, a glass plate is used for the transparent flat plate 21, but a quartz glass plate, which is a non-fluorescent material, is preferable in consideration of a case where the transparent flat plate 21 is used for observing fluorescence generated by irradiating a sample with excitation light. .

The anti-reflection film 22 is formed on one surface of the transparent flat plate 21 and has a refractive index and a film thickness of each of the transparent flat plate 21 and the air layer such that the reflectance for light of a predetermined wavelength is reduced. Appropriately designed according to the refractive index. Here, the predetermined wavelength is an excitation light wavelength when used for fluorescence observation, although it depends on the use of the cover glass 20.
The anti-reflection film 22 may be formed on one entire surface of the transparent flat plate 21 as shown in this figure, or may be formed on a part thereof, for example, formed only in the central region of the surface. It may be.

As described above, the antireflection film 22 is formed of the transparent flat plate 21.
However, it is not easy to identify the surface of the transparent flat plate 21 on which the antireflection film 22 is formed. Therefore, an identification mark 23 is provided to facilitate the identification. The surface on which the identification mark 23 is written is the transparent flat plate 2.
It may be a surface on which one antireflection film 22 is formed,
The surface on the opposite side may be used.
2 may be on. The position where the identification mark 23 is written is
The periphery of the transparent flat plate 21 is preferable so as not to hinder the sample observation. Further, the identification mark 23 may be a character, a graphic, a symbol, a trademark, a logo of a maker name or any other mark, and may be displayed by not only printing but also by engraving.

Since the cover glass has a small thickness, it is difficult to determine on which side of the transparent flat plate 21 the identification mark 23 is written, and therefore, when viewed from one side. It is preferable to adopt an identification mark 23 having a shape different from each other when viewed from the other side.

Next, the usage of the cover glass according to the present invention will be described. FIG. 4 is an explanatory diagram of the use of the cover glass according to the present invention in a fluorescence microscope.

In this figure, a conventional slide glass 10A is shown.
The sample 30 is sandwiched between the surface of the cover glass 20 according to the present invention on which the antireflection film 22 is not formed, and the sample 30 is placed on a sample stage (not shown). Is irradiated on the sample 30 and the fluorescence generated in the sample 30 is observed.

In this case, when the sample 30 is irradiated with the excitation light A, the reflected light B1
Is generated, and reflected light B3 is generated at the interface between the conventional slide glass 10A and the air layer, and reflected light B4 is generated at the interface between the condenser lens 50 and the air layer. However, reflected light is reduced at the interface between the cover glass 20 and the air layer according to the present invention. Since this boundary surface is at a position close to the objective lens 40, the reduction of the reflected light greatly reduces the auto-fluorescence.

In FIGS. 2 and 4, each of the embodiments using only one of the slide glass and the cover glass according to the present invention has been described. However, it is possible to use both the slide glass and the cover glass according to the present invention at the same time. For example, the reflected light of the excitation light A incident on the objective lens 40 is further reduced, and thus the autofluorescence is further reduced. Further, when the slide glass according to the present invention is used, the efficiency of irradiating the sample 30 with the excitation light A is improved, and the fluorescence intensity is increased, so that the S / B ratio is also improved from this point.

In the above embodiment, the slide glass and the cover glass suitable for use with the bright-field illumination fluorescence microscope have been described. In this case, since the excitation light A is incident on the slide glass and the cover glass substantially perpendicularly, the antireflection film is designed so that the reflectance is reduced under the incident conditions.

Next, a slide glass and a cover glass suitable for use with a dark-field illumination fluorescence microscope will be described. In this case, the excitation light A does not vertically enter the sample 30 or the like, but obliquely enters. FIG. 5 is an explanatory diagram of excitation light irradiation and fluorescence detection in a dark-field illumination fluorescence microscope.

As shown in this figure, in the dark-field illumination fluorescence microscope, the excitation light A is made into an annular luminous flux, then enters the annular-shaped dark-field condenser lens 60, and
The light is converged and illuminated to zero. Then, the fluorescent light B generated in the sample 30 is condensed by the dark field objective lens 70 coaxially arranged inside the dark field condenser lens 60, and guided to a detector (not shown). As described above, the excitation light A is obliquely incident on the cover glass 20 and the slide glass 10.

By the way, in general, the reflectance when light is vertically incident on a glass plate having a refractive index of 1.5 is about 4%, while the reflectance when obliquely incident is large. For example, the numerical aperture of the dark field condenser range 60 is
0.8 to 0.99 (0.88 to 0.9 in a practical range)
8), the range of the incident angle of the excitation light A (the angle between the straight line perpendicular to the surface of the cover glass and the incident direction) is:
It is in the range of 53 ° to 82 ° (62 ° to 79 ° in a practical range). When the S-polarized excitation light A enters the glass plate at such an incident angle, the reflectance is 13% to 61%.
(In a practical range, 19% to 51%). The average value of the reflectance of each of the S-polarized light and the P-polarized light is 6.
The range is 4% to 46% (10% to 35% in a practical range).

As described above, when the excitation light A is obliquely incident, the reflectivity of the conventional cover glass becomes extremely large, so that the efficiency of irradiating the sample 30 with the excitation light A decreases and the intensity of the fluorescent light C also decreases. As a result, the S / B ratio deteriorates. Further, the reflectance of the conventional slide glass becomes extremely large, and the fluorescence generated by irradiating the sample 30 with the excitation light A reflected by the slide glass becomes noise, which also causes a reduction in the S / B ratio. Become. Further, since the reflectance of the S-polarized light and the P-polarized light for the excitation light A is different, the S / B ratio is different depending on the polarization state of the excitation light A.

Therefore, the slide glass 10 and the cover glass 20 according to the present invention used together with the dark-field epi-illumination microscope as described above have the above-mentioned incident angles of 53 ° to 82 ° (63 ° to 79 ° in a practical range). It is preferable to reduce the reflectance in the range of ゜). Then, as shown in the figure, the side on which the anti-reflection film 22 of the cover glass 20 is formed is used as the upper side (the side on which the excitation light A is incident), and the side of the slide glass 10 on which the anti-reflection film 12 is formed is set as the lower side. By using this, the efficiency of irradiating the sample 30 with the excitation light A is improved, and the fluorescence that becomes noise generated by the irradiation of the excitation light A reflected by the slide glass 10 is reduced, so that the S / B ratio is excellent. Fluorescence measurement becomes possible. Further, the dependency of the S / B ratio on the polarization state of the excitation light A is reduced.

In FIG. 5, the embodiment in which both the slide glass and the cover glass according to the present invention are used at the same time has been described. However, in the case where only one of the slide glass and the cover glass according to the present invention is used. Even so, it is effective for improving the S / B ratio.

In the description of the above embodiment, the slide glass and the cover glass have been described on the assumption that they are used for a fluorescence microscope. However, the slide glass and the cover glass are also suitably used for other optical microscopes. The refractive index and the film thickness are designed so that the reflectance is small with respect to the illumination light used in the optical microscope.

[0044]

According to the present invention, as described in detail above, each of the slide glass and the cover glass
The transparent flat plate has an antireflection film on one or all of one surface thereof, and has an identification mark for identifying one of the surfaces. In particular, when each of the slide glass and the cover glass is used together with a fluorescence microscope, the antireflection film is designed to reduce the reflectance with respect to the excitation light, and the transparent flat plate is a quartz glass plate.

By doing so, the reflectance on the surface of the transparent flat plate on which the anti-reflection film is formed is reduced, so that the amount of reflected light of the excitation light incident on the objective lens is reduced, and the amount of light generated in the objective lens is reduced. Autofluorescence is also reduced, thus improving the S / B ratio in sample observation. Further, the identification of the surface of the transparent flat plate on which the antireflection film is formed is facilitated by the identification mark.

[Brief description of the drawings]

FIG. 1 is a perspective view of a slide glass according to the present invention.

FIG. 2 is an explanatory view of the use of the slide glass according to the present invention in a fluorescence microscope.

FIG. 3 is a perspective view of a cover glass according to the present invention.

FIG. 4 is a diagram illustrating the use of the cover glass according to the present invention in a fluorescence microscope.

FIG. 5 is an explanatory diagram of excitation light irradiation and fluorescence detection in a dark-field illumination fluorescence microscope.

FIG. 6 is an explanatory diagram of excitation light reflection in a fluorescence microscope.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 10 ... Slide glass, 11 ... Transparent flat plate, 12 ... Anti-reflective film, 13 ... Identification sign, 20 ... Cover glass, 21 ... Transparent flat plate, 22 ... Anti-reflection film, 23 ... Identification sign, 30 ... Sample, 40 ... Objective lens , 50 ... condenser lens, 60
... dark field condenser lens, 70 ... dark field objective lens,
A: excitation light, B1, B2, B3, B4: reflected light, C: fluorescence.

Continued on the front page (72) Inventor Shuji Toyonaga 5000 Hiraguchi, Hamakita City, Shizuoka Prefecture Inside Molecular Biophotonics Laboratory Co., Ltd.

Claims (10)

[Claims] 1. A slide glass having an antireflection film on the whole or a part of one surface of a transparent flat plate, and having an identification mark for identifying the one surface. 2. The slide glass according to claim 1, wherein the antireflection film reduces a reflectance with respect to excitation light. 3. The slide glass according to claim 1, wherein said transparent flat plate is a quartz glass plate. 4. The slide glass according to claim 1, wherein the antireflection film reduces a reflectance with respect to light that is incident substantially perpendicularly. 5. The anti-reflection film has an incident angle of 53 ° or more and 8 or more.
2. The slide glass according to claim 1, wherein the reflectance is reduced for light incident in a range of 2 [deg.] Or less. 6. A cover glass having an antireflection film on all or a part of one surface of a transparent flat plate and having an identification mark for identifying one surface thereof. 7. The cover glass according to claim 6, wherein the antireflection film reduces a reflectance with respect to excitation light. 8. The cover glass according to claim 6, wherein said transparent flat plate is a quartz glass plate. 9. The cover glass according to claim 6, wherein said anti-reflection film reduces the reflectance with respect to substantially perpendicularly incident light. 10. The cover glass according to claim 6, wherein said anti-reflection film reduces the reflectance with respect to light incident at an incident angle of not less than 53 ° and not more than 82 °.