JPH09292572A - Vertical illumination type fluorescence microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently use excitation light irradiating a sample and to obtain a bright fluorescence image by providing a reflection surface so that the excitation light transmitted through the sample may be restored to the sample again. SOLUTION: The excitation light from a light source 1 is formed into an image by a collector lens 2, transmitted through a relay lens 4, reflected by a dichroic mirror 5, and made incident on an objective lens 6 functioning also as a condenser lens. After it is formed into the image again at the pupil position of the lens 6, it illuminates the sample 8 placed on a slide glass 9. The sample 8 receives the excitation light and emits fluorescence, and then the fluorescence is transmitted through the lens 6 and the mirror 5 and formed into the fluorescence image 10. Furthermore, after the excitation light illuminates the sample 8, it is reflected by the reflection surface 9a of the slide glass 9, made incident on the sample 8 again and illuminates the sample. The fluorescence arriving at the lens 6 out of the fluorescence excited by the excitation light which goes and returns and emitted from the sample 8 is formed into the fluorescence image 10.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fluorescence microscope,
Particularly, it relates to an epi-illumination type fluorescence microscope.

[0002]

A fluorescence microscope has been widely used for examining the characteristics of a sample by irradiating the sample with excitation light and observing fluorescence emitted from the sample and having a wavelength longer than that of the excitation light. Fluorescent microscopes include a transmission type that uses a condenser lens and an epi-illumination type that uses the objective lens itself as an illumination system depending on the way of irradiating the excitation light. At present, the epi-illumination type is the mainstream, and the illumination light emitted from the objective lens that also serves as a condenser lens illuminates and transmits the sample, and the excitation light after that is not particularly used.

It is difficult to obtain a bright fluorescent image with a fluorescence microscope. This is a sample of 360nm, 400nm,
This is because various short wavelengths such as 440 nm are irradiated as excitation light and weak fluorescence from the sample is observed. As described above, since the amount of fluorescence emitted from the sample is weak, the exposure time at the time of taking a photograph is long, and it is easily affected by vibration. Therefore, efforts have been made to obtain bright fluorescent images by various optical considerations. For example, to use a high-intensity mercury lamp that emits strong excitation light as a light source, or to efficiently take in fluorescence from a sample,
The numerical aperture (NA) of the objective lens of the imaging system is increased. However, it is still difficult to say that a fluorescent image with sufficient brightness has been obtained. Therefore, an object of the present invention is to provide an epi-illumination fluorescence microscope that can efficiently use excitation light that illuminates a sample and obtain a bright fluorescence image.

[0004]

The present invention has been made to solve the above-mentioned problems, that is, in a reflection-type fluorescence microscope, a reflecting surface is provided so that the excitation light transmitted through the sample returns to the sample again. It is an epi-illumination fluorescence microscope characterized by being provided.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a first embodiment of the present invention, in which a light source 1
The excitation light from is imaged by the collector lens 2, and the aperture stop 3 is arranged at the imaging position. The excitation light that forms the light source image further passes through the relay lens 4, is reflected by the dichroic mirror 5, enters the objective lens 6 that also serves as a condenser lens, and is re-imaged at the pupil position of the objective lens 6, The sample 8 is illuminated. This configuration is Koehler illumination.

A sample 8 is placed on a slide glass 9, and a cover glass 7 covers the sample 8. The sample 8 receives the excitation light and emits fluorescence, and the fluorescence passes through the objective lens 6 and the dichroic mirror 5 and forms a fluorescence image 10. The dichroic mirror 5 is formed so as to reflect excitation light having a short wavelength and transmit fluorescence having a long wavelength. As shown in FIG. 2, a surface 9a of the slide glass 9 on the sample 8 side is coated with a vapor deposition film having a characteristic of reflecting excitation light having a short wavelength and transmitting or absorbing fluorescence having a long wavelength. .

This embodiment is constructed as described above,
After the excitation light is incident on the sample 8 to illuminate the sample, the excitation light is reflected by the reflecting surface 9a of the slide glass 9 and is incident on the sample 8 again to illuminate the sample. After that, the excitation light passes through the objective lens 6 and is reflected by the dichroic mirror 5,
The excitation light does not reach the fluorescent image 10. Fluorescence is emitted from the sample 8 by being excited by the reciprocating excitation light, and of the fluorescence, the fluorescence reaching the objective lens 6 forms a fluorescence image 10. Further, among the fluorescence, the fluorescence reaching the reflection surface 9a of the slide glass 9 does not deteriorate the contrast of the fluorescent image 10 because the reflectance of the vapor deposition film of the slide glass 9 is low.

Thus, according to the present embodiment, the sample 8 is excited by the excitation light for two round trips, so that the brightness is about twice as high as that of the conventional example which is excited only by the excitation light on the outward path. A fluorescent image 10 can be obtained. Although the reflective surface 9a is formed on the surface of the slide glass 9 on the side of the sample 8 in this embodiment, the surface on the opposite side of the sample 8 can be used as the reflective surface. It is also possible to provide a reflecting surface separately from 9.

Next, FIG. 3 shows a second embodiment. In this embodiment, a reflecting surface is not provided on the slide glass 9, and a reflecting surface is provided below the slide glass 9 as a separate body from the slide glass 9. And a concave mirror 11 whose center of curvature is approximately at the position of the sample 8 is used as the reflecting surface. With such a configuration, the same effect as that of the first embodiment can be obtained.

[0010]

As described above, according to the present invention, only by adding simple elements to the conventional epi-illumination type fluorescence microscope, the irradiation efficiency can be improved about twice as compared with the conventional illumination method. ,
Therefore, a bright fluorescent image can be obtained.

[Brief description of drawings]

FIG. 1 is a configuration diagram showing a first embodiment of the present invention.

FIG. 2 is a diagram showing reflectance characteristics of a reflective surface of a slide glass.

FIG. 3 is a configuration diagram showing a second embodiment.

[Explanation of symbols]

 1 ... Light source 2 ... Collector lens 3 ... Aperture stop 4 ... Relay lens 5 ... Dichroic mirror 6 ... Objective lens 7 ... Cover glass 8 ... Sample 9 ... Slide glass 9a ... Reflecting surface 10 ... Fluorescent image 11 ... Concave mirror

Claims (5)

[Claims] 1. An epi-illumination fluorescence microscope, wherein a reflection surface is provided in the epi-illumination fluorescence microscope so that the excitation light transmitted through the specimen returns to the specimen again. 2. The epi-illumination fluorescence microscope according to claim 1, wherein the reflecting surface is formed on a slide glass on which a sample is placed. 3. The epi-illumination fluorescence microscope according to claim 1, wherein the reflecting surface is arranged at a position for reflecting the excitation light transmitted through the slide glass on which the sample is placed. 4. The epi-illumination fluorescence microscope according to claim 3, wherein the reflecting surface is formed by a concave mirror having a center of curvature substantially at the position of the sample. 5. The epi-illumination fluorescence microscope according to claim 1, wherein the reflectance characteristic of the reflecting surface is such that the reflectance for excitation light is higher than the reflectance for fluorescence.