JP2002196244A - Microscope and illumination light antireflection unit for microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent microscope which is capable of effectively lessening the reflection of exciting light. SOLUTION: This microscope includes a stage 17, an objective lens 14, an irradiation optical system for irradiating observation objects 15 and 18 with illumination light through the objective lens 14 and the illumination light antireflection unit 200. The illumination light antireflection unit 200 includes lenses 54 and 55 for converging the exciting light past the observation objects 15 and 18 and a deflecting member 55 for deflecting the exciting light past the lenses 54 and 55.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a fluorescence microscope.

[0002]

2. Description of the Related Art For example, when a fluorescence image is observed with a fluorescence microscope, a specimen is usually irradiated with excitation light through an objective lens. The sample irradiated with the excitation light causes a Stoke shift in a material constituting the sample, and emits fluorescence having a longer wavelength than the excitation light. Fluorescence observation can be performed by forming an image of the fluorescence with an objective lens and observing the image. As the excitation light, light in a wavelength range from ultraviolet to blue is often used.

[0003] The intensity of the fluorescence emitted from the specimen is very small as compared with the intensity of the excitation light, and is generally only about 1 / 100,000 of the intensity of the excitation light. For this reason, in the case where the wavelength of the excitation light includes the visible region, when the reflected light of the excitation light from various members enters the objective lens, the contrast of the fluorescent image is reduced and the image quality is reduced. The factors that cause the reflected light of the excitation light include reflection of the excitation light by a stage or a slide glass, and reflection of the excitation light by a lens such as a condenser lens for transmission illumination disposed below the stage.

Therefore, a fluorescence microscope has been proposed which suppresses the reflection of excitation light by disposing an antireflection plate having a low reflectance under the stage. This configuration will be specifically described with reference to FIG. The microscope in FIG. 2 includes an objective lens 31, a stage 34, a condenser lens 35 for transmitted illumination, and a lens 36 for transmitted illumination on an optical axis 100 of the objective lens 31 in this order. At the lower part of the stage 34, a black flat plate 30 with low reflectance is attached. At the time of fluorescence observation, excitation light is applied to the specimen 32 and the slide glass 33 on the stage 34 through the objective lens 31. The excitation light passes through the sample 32 and the slide glass 33 and passes through the opening of the stage 34, but is applied to the flat plate 30, and the excitation light is directly transmitted to the condenser lens 35 and the transmitted illumination lens 3
It does not reach up to 6. Since the reflectance of the flat plate 30 is low, the amount of reflected light returning to the objective lens 31 can be reduced as compared with the case where the excitation light is reflected by the condenser lens 35 and the transmitted illumination lens 36.

As another configuration, there has been proposed a configuration in which a condenser lens is removed during fluorescence observation and a cylinder 42 is mounted on the condenser receiving portion 40 in place of the condenser lens as shown in FIG. A black cloth 41 having a low reflectance is bonded to the inner surface of the cylinder 42. Stage 3
The excitation light that has passed through the opening 4 is reflected by the cloth 41 having a low reflectance, so that the excitation light is not reflected by the condenser lens 35 or the transmitted illumination lens 36 than by the objective lens 3.
The amount of reflected light returning to 1 can be reduced.

[0006]

The configuration in which the black anti-reflection plate is placed under the stage as shown in FIG. 2 has the advantage that the anti-reflection plate can be easily mounted. Since there is a limit in reducing the rate, it is inevitable that reflected light returns to the objective lens with a certain intensity. The antireflection plate of FIG. 2 has a numerical aperture (N.
A. When the excitation light from the large objective lens is received, scattered light is easily generated, and the effect of preventing reflection is reduced.

The arrangement of FIG. 3 also has the advantage that the tube can be easily mounted, and that the excitation light from the objective lens having a relatively large numerical aperture can be cut off. However, there is a limit in reducing the reflection of the excitation light passing on the optical axis that easily affects the fluorescent image.

It is an object of the present invention to provide a microscope capable of effectively reducing the reflection of illumination light.

[0009]

In order to achieve the above object, the present invention provides the following fluorescence microscope.

That is, a stage for mounting an observation object, an objective lens, an irradiation optical system for irradiating illumination light to the observation object through the objective lens, and the objective lens with the stage interposed therebetween. An illumination light anti-reflection unit disposed at a position facing the light source, wherein the illumination light anti-reflection unit comprises one or more lenses for converging the illumination light having passed through the observation object, and the one or more lenses. A deflecting member for deflecting the illumination light passing through the lens.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A fluorescence microscope according to one embodiment of the present invention will be described with reference to the drawings.

As shown in FIG. 1, a fluorescence microscope according to the present embodiment has an epi-illumination device 12 and a revolver 21 mounted on a main body 114.
1 and a sub-stage 210. The lamp house 11 is connected to the epi-illumination device 12. On the epi-illumination device 12, a lens barrel 17 and an eyepiece 16 are provided.
Are installed in order. A plurality of objective lenses 14 can be attached to the revolver 211.

The sub-stage 210 includes a vertical movement mechanism 212
Is attached to the main body 114 via the. The vertical movement mechanism 212 includes a handle 121, and the handle 121
Is rotated to move the sub-stage 210 to the main body 1.
14 can be moved up and down. The stage 17 having the opening 17a (see FIG. 4) is mounted on the sub-stage 210. On the stage 17, a specimen 15 placed on a slide glass 18 is arranged as an observation target.

A unit receiver 10 is arranged below the stage 17. The unit receiving section 10
It is supported by the substage 210 via a vertical movement mechanism 310. Therefore, the handle 3 of the vertical movement mechanism 310
By rotating 10a, unit receiving portion 10 can be moved up and down with respect to substage 210. An excitation light antireflection unit 200 or a condenser lens unit (not shown) for transmitted illumination is detachably mounted on the unit receiving portion 10. The unit receiving portion 10 when the unit 200 is not mounted has a shape as shown in FIG.

At the base of the main body 114, a transmitted light lens 20 used for transmitted illumination is arranged.

In the epi-illumination device 12, a filter block 13 is arranged. The filter block 13 includes an excitation filter 13a for transmitting only excitation light having a desired wavelength, a dichroic mirror 13b, and a barrier filter 13c.

The objective lens 1 selected by the lens barrel 17, the filter block 13 of the epi-illumination device 12, and the revolver 211
4. The stage 17, the unit 200 supported by the unit receiver 10, and the transmitted light lens 20 are all located on the optical axis 100.

Here, the structure of the excitation light reflection preventing unit 200 will be described with reference to FIGS. The excitation light antireflection unit 200 includes a first lens 54 and a second lens 5.
5, an optical element mirror 56, and a housing 58. First lens 54, second lens 55, and mirror 56
Means that the excitation light antireflection unit 200 is
When mounted on the optical axis 100, they are arranged on the optical axis 100 in order from the specimen 15 side. First lens 54
Is formed in a plane such that the lens surface 54 a on the specimen 15 side is parallel to the lower surface of the slide glass 18 on the stage 17, and is fixed to the opening at the top of the unit 200. Also, the lower lens surface 54b of the first lens 54
Is formed into a curved surface in order to collect and emit the light incident on the first lens 54 from the sample 15 side. The second lens 55 is configured to further condense the light incident from the first lens 54 and emit the light toward the mirror 56. The second lens 55 is fixed by a fixing member (not shown) provided on the inner wall of the unit 200. It is supported on the lens end face. Mirror 56
Is directed to deflect the light from the second lens 55 perpendicularly to the optical axis 100, and is supported on the mirror end surface by a fixing member (not shown) provided on the inner wall of the unit 200. The housing 58 is provided with a window 57 for passing the light deflected by the mirror 56.

In this embodiment, a liquid 53 such as water is placed on the lens surface 54a of the first lens 54, and the liquid 53 fills the space between the first lens 54 and the slide glass 18. Is preferred. The liquid 53 is
The refractive index of the slide glass 18 or the first lens 5 is higher than that of air.
Use something close to 4. The refractive index of the liquid 53 is most preferably the same as the refractive index of the slide glass 18 and the first lens 54. However, even if they are not completely the same, a sufficient antireflection effect of the excitation light can be obtained.
In addition, the liquid is selected in consideration of the refractive index of the first lens 54. Here, water is used. The lens surface 54a is
Here, the plane is a plane, but the plane is not necessarily required to be a plane, and may be a concave plane.

The slide glass 18 and the first lens 5
The liquid 53 is not necessary if the device 4 is installed in close contact with no gap. At this time, it is preferable that the refractive indexes of the slide glass 18 and the first lens are substantially equal.

The procedure for observing a fluorescent image of a specimen by epi-illumination with the fluorescent microscope of this embodiment and the operation of each section will be described. First, the excitation light reflection preventing unit 200 is mounted on the unit receiving portion 10 as shown in FIGS. At this time, the unit receiving portion 10 is kept down near the base portion of the microscope main body. That is, a space is created between the lower surface of the stage 17 and the lens surface of the first lens 54. On the stage 17, a specimen 15 placed on a slide glass 18 is mounted. Next, a liquid (here, water) 53 is dropped on the lens surface 54a of the first lens 54 of the excitation light antireflection unit 200, and the droplet is raised on the lens surface 54a by the surface tension of the liquid 53. The liquid 53 may be dropped before the slide glass 18 is mounted on the stage 17. In this state, operating the handle 310a,
The excitation light anti-reflection unit 200 is moved closer to the stage 17 so that the upper surface of the droplet contacts the lower surface of the slide glass 18. As a result, the space between the first lens 54 and the slide glass 18 is filled with the liquid 53 by capillary action.

Next, the stage 17 is moved up and down by operating the handle 121 to move the sample 15 to the focal position of the objective lens 14. At this time, since the unit receiving portion 10 also moves integrally with the stage 17, the distance between the first lens 54 and the slide glass 18 is maintained, and the space between the first lens 54 and the slide glass 18 remains filled with the liquid 53. is there.

In this state, the excitation light is emitted from the lamp house 11. Here, a mercury lamp having an emission wavelength of 350 to 1000 nm is used as the lamp house 11. The emitted excitation light travels along the optical axis 101 in FIG. 1 and enters the filter block 13. In the filter block 13, only the excitation light having a desired wavelength (for example, 380 to 420 nm) passes through the excitation filter 13a and is deflected in the direction of the optical axis 100 by the dichroic mirror 13b. The polarized excitation light 14 passes through the objective lens 14 and passes through the specimen 15
Is irradiated. The irradiation of the excitation light 14 causes the specimen 15
Fluoresces. The emitted fluorescence is reflected by the objective lens 14.
After being condensed by the filter, the light passes through the dichroic mirror 13b and passes through the barrier filter 13c. Then, the light is deflected by the lens barrel 17 to form an image, and reaches the eyepiece 16. Thereby, a fluorescent image can be observed.

On the other hand, the excitation light applied to the sample 15 passes through the sample 15, passes through the slide glass 18, and enters the first lens 54 of the excitation light antireflection unit. At this time, since the space between the slide glass 18 and the first lens 54 is filled with the liquid 53, the slide glass 1
The interface between the liquid 8 and the liquid 53 and the interface between the liquid 53 and the first lens 54 have almost no difference in refractive index. For this reason, the excitation light can pass through the first lens 54 without being substantially reflected at these interfaces. The excitation light incident on the first lens 54 is
The light is converged when emitted from b, and is condensed on the second lens 55. After being further converged by the second lens 55, the excitation light is deflected by the mirror 56 in a direction perpendicular to the optical axis 100. The deflected excitation light passes through the window 57 and exits to the outside. As described above, it is preferable that the window 57 is provided in the unit to release the deflected excitation light to the outside. However, the window 57 may not be provided, and a light absorbing member may be provided on the inner wall of the unit to which the excitation light is applied. In this case as well, since the excitation light is reduced by the first lens 54 and the second lens 55, the reflected light can be reduced more effectively than the conventional one shown in FIG.

Therefore, the excitation light incident on the slide glass 18 can be made incident on the first lens 54 without being reflected, and most of the excitation light incident on the slide glass 18 can be reflected by the first lens 54 and the second lens 55. To converge, and can be made to fall out in a direction different from that of the objective lens 14. Thereby, the reflected light of the excitation light incident on the objective lens 14 can be effectively reduced, and the deterioration of the image quality of the fluorescent image can be prevented. Also, instead of the mirror 56,
An optical element having a dimming function, such as an ND filter, or a prism can also be used.

Further, since the excitation light antireflection unit 200 of the present embodiment uses the liquid 53, it is a lens system capable of converging almost all of the excitation light without reflecting it, but has a configuration. It also has the advantage of being simple.

Further, by shifting the optical axis of the first lens 54 with respect to the optical axis 100 of the objective lens 14, the optical axis 10
The reflected light from the first lens 54 incident on the objective lens 14 along 0 can be further reduced. With such a configuration, it is possible to reduce reflected light by arranging, for example, an antireflection cloth or the like instead of the mirror 565.

In the fluorescence microscope of this embodiment, during observation by transmitted illumination, the excitation light reflection preventing unit 200 is detached from the unit receiving portion 10 so as to be in a state as shown in FIG. 5, and then a condenser lens unit (not shown). ) Allows observation with transmitted light as before. Further, the condenser lens unit is removed from the conventional fluorescence microscope, and the excitation light anti-reflection unit 200 of the present application is removed.
The configuration of the fluorescence microscope according to the present embodiment can be realized only by attaching.

In the present embodiment, a fluorescence microscope has been described as an example. However, the present invention is not limited to this, and the present invention can be applied to a microscope that illuminates a sample and reflects light from the sample.

The illumination light (excitation light) antireflection unit 200 can be mounted on the unit receiving portion 10 instead of the condenser lens 35 as described above. This is the same as the method attached to the unit 10. Specifically, a male screw is inserted into one or more holes formed in the receiving portion 10, and the illumination light (excitation light) antireflection unit 200 mounted on the receiving portion 10 is pressed by the male screw to be attached. It is possible.

The shape of the first lens 54 of the illumination light (excitation light) anti-reflection unit 200 is preferably a dome shape which is semicircular when viewed from the side. Also, the first
Considering the ease of mounting the lens 54 on the housing 58, it is preferable that the contact portion with the housing 58 be linear.

In the above-described embodiment, one condenser lens unit attached to the unit receiving portion 10 is removed, and the illumination light (excitation light) antireflection unit 200 is attached. It is not limited. For example, the unit receiving portion 10
With a turret structure that allows a plurality of condenser lenses to be mounted, and a configuration in which a condenser lens and an illumination light (excitation light) antireflection unit are mounted on the turret, it is only necessary to rotate the turret to align the optical axis. In addition, the condenser lens and the illumination light (excitation light) antireflection unit can be exchanged. In addition to the turret, a block in which a plurality of condenser lens mounting parts (mounting holes) are formed is used. A condenser lens and an anti-reflection unit are mounted on this block, and the block is slid with respect to the optical axis for positioning. May be performed.

[0033]

As described above, according to the present invention, it is possible to provide a microscope capable of effectively reducing the reflection of illumination light.

[Brief description of the drawings]

FIG. 1 is a side view showing an overall configuration of a fluorescence microscope according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view showing a configuration around a stage of a conventional fluorescence microscope.

FIG. 3 is a cross-sectional view showing a configuration around a stage of a conventional fluorescence microscope.

FIG. 4 is a sectional view taken along the line AA ′ showing a configuration around a stage of the fluorescence microscope of FIG. 1 of the present invention.

FIG. 5 is a partial side view showing a state in which the antireflection unit is removed from the unit supporter 10 in the fluorescence microscope of FIG. 1 of the present invention.

6 is a cross-sectional view taken along the line BB 'showing a configuration around the stage of the fluorescence microscope of FIG. 1 of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 10 ... Unit receiving part, 11 ... Lamp house, 12 ... Epi-illumination device, 13 ... Filter block, 13a ... Excitation filter, 13b ... Dichroic mirror, 13c ... Barrier filter, 14 ... Objective lens, 15 ... Sample, 16 ... Eyepiece Reference numeral 17: lens barrel, 18: slide glass, 20: lens for transmitted light, 30: flat plate for anti-reflection, 31: objective lens, 32: specimen, 33: slide glass, 34: stage, 35: condenser lens, 36 ... Lens for transmitted light,
40: capacitor receiving portion, 41: anti-reflection cloth, 42:
Anti-reflection cylinder, 53 liquid, 54 first lens, 55
2nd lens, 56 mirror, 57 window, 58 housing, 2
00: Excitation light anti-reflection unit, 310: Vertical movement mechanism,
310a ... handle, 211 ... revolver.

Claims (4)

[Claims] 1. A stage for mounting an observation object,
An objective lens, an illumination optical system for irradiating the observation object with illumination light through the objective lens, and an illumination light antireflection unit disposed at a position facing the objective lens with the stage interposed therebetween. The illumination light antireflection unit includes one or more lenses for converging the illumination light that has passed through the observation target,
A deflecting member for deflecting the illumination light passing through the one or more lenses. 2. The microscope according to claim 1, wherein
The lens surface of the above lens closest to the observation object is
A microscope formed to have a shape on which a liquid can be placed in order to hold the liquid between the lens surface and the observation object. 3. The microscope according to claim 1, wherein the support unit supports the illumination light anti-reflection unit.
In order to adjust the distance between the illumination light anti-reflection unit and the stage, it has a moving mechanism for moving the support portion, and the illumination light anti-reflection unit is detachable from the support portion. A microscope characterized by the above-mentioned. 4. A condenser lens for transmitting illumination of a microscope, wherein the illumination light has passed through an observation object, and one or more lenses for converging the illumination light; a deflecting member which deflects the illumination light having passed through the one or more lenses; An anti-reflection unit for illumination light which can be attached to a microscope in place of a condenser lens for transmitted illumination, comprising an attachment portion for attachment to a part.