JPH0868756A - Vertical fluorescence illumination device of microscope - Google Patents

Abstract

PURPOSE: To irradiate only a part of an observation visual field region with breaking light for breaking a caged compound by providing a visual field iris prescribing the irradiation region of breaking light to a breaking light irradiation optical system. CONSTITUTION: The light from a breaking light irradiation light source 16 propagates through an optical fiber 18 to reach an emitting end 18b to be emitted as a secondary light source. This light illuminates a sample 1 through an objective lens 20 to break the caged compd. contained in a sample 1. The irradiation region with breaking light is prescribed on the sample l by a visual field iris FS2. The fluorescence generated from the sample irradiated with the exciting light from an exciting light irradiation light source 11 or the breaking light from the light source transmits through the dichroic mirror D in a relay lens system 3 and is reflected by a mirror 4 to form a magnified image 5. The light from the magnified image 5 is again formed into an image 8 through a relay lens system 6 and a prism 7. The image 8 is observed by the naked eye 10 of an observer through an eyepiece 9.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an epi-illumination device for a microscope.

[0002]

2. Description of the Related Art As a microscope observing method, there is an epifluorescence method in which short-wavelength excitation light from a light source such as a mercury lamp is illuminated on an observation visual field region on a sample, and long-wavelength fluorescence emitted from the sample is observed. Specifically, the dichroic mirror arranged in the parallel light beam path of the observation optical system from the objective lens to the magnified image reflects excitation light of a short wavelength from a mercury lamp or the like, and the reflected light is transmitted through the objective lens to the specimen. To illuminate. A caged compound method using a caged compound is known as one method of this epifluorescence method.

The principle of the caged compound method will be described below. Generally, it is considered that the action of cells in the living body is regulated by calcium. Then, the caged compound is such that, for example, this calcium molecule is taken into a cage by another molecule (caged). This caged compound is destroyed by strong light energy and releases calcium. At this moment, the living body causes movement by calcium.
The caged compound method attempts to capture the movement of cells at this time.

Calcium released by breaking the caged compound emits specific fluorescence, and therefore an epi-illumination illuminator is used to observe the movement of calcium. In this case, a light source is needed to destroy the caged compound. The caged compound method is a method in which the epifluorescence method and the caged compound are combined. The compound used in the caged compound method is not limited to a caged compound that releases calcium, and a caged compound that releases nucleotides, neurotransmitters, fluorescent dyes, luciferin and the like may be used.

In the conventional epi-illumination illuminator for a microscope using the caged compound method, it is necessary to irradiate the specimen with strong light for destroying the caged compound, that is, destruction light. Generally, destructive light can be obtained by applying a high voltage to, for example, a xenon light source. The destructive light thus obtained is directly applied to the entire specimen via a light guide such as an optical fiber.

[0006]

In the conventional epi-illumination illuminator for a microscope as described above, the entire specimen is directly illuminated with destructive light via an optical fiber or the like. Therefore, the irradiation area of the destructive light cannot be narrowed down to a specific area of the observation visual field area. As a result, there is a disadvantage in that it is not possible to meet the demand for observing only the reaction in a specific part of the cell during observation.

Further, in the conventional epi-illumination illuminator for a microscope, the entire sample is directly illuminated with the destructive light, so that the light is dispersed. For this reason, there is a disadvantage in that it is difficult to obtain a breaking light having an intensity sufficient to break the caged compound. The present invention has been made in view of the above-mentioned problems, and it is possible to irradiate only a partial region of the observation visual field with the destructive light for destructing the caged compound, and the epi-fluorescent illumination device of the microscope. The purpose is to provide.

[0008]

In order to solve the above problems, in the present invention, an excitation light irradiation optical system for irradiating a sample with excitation light for epi-illumination, and a caged compound contained in the sample. With a destructive light irradiation optical system for irradiating the specimen with destructive light for destructing the, in the epi-illumination fluorescence illumination device of the microscope, the destructive light irradiation optical system, and A condensing lens means for condensing light from the light source into an observation visual field area of the specimen, and an irradiation area of the destructive light on the specimen positioned between the light source and the condensing means. Provided is an epi-illumination illuminator which is provided with a field stop for defining.

According to a preferred aspect of the present invention, the field diaphragm is narrowable. Further, the field stop is
It is preferably movable in a plane perpendicular to the optical axis. Further, it is preferable that the light source is arranged in the vicinity of the field stop and is movable integrally with the field stop in a plane perpendicular to the optical axis.

[0010]

In the epi-illumination illuminator of the present invention, the destructive light for destructing the caged compound contained in the specimen is condensed on the specimen by the condenser lens means via the field stop. Therefore, it is possible to irradiate the destructive light only to a part of the observation visual field region of the sample to which the excitation light for epi-illumination is irradiated, and observe the reaction of a part of the cells of the sample.

By making it possible to narrow down the field stop, it is possible to irradiate a region of a desired size with destructive light of a desired intensity. In addition, by making the field stop movable two-dimensionally in a plane perpendicular to the optical axis, fluorescent light can be observed over the entire observation visual field area while sequentially irradiating destructive light to any area of the observation visual field area. It can be carried out.

[0012]

Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 is a diagram showing the configuration of an epi-illumination device for a microscope according to a first embodiment of the present invention. The illustrated microscope is an inverted microscope, and an objective lens 2 for epi-illumination is arranged below the specimen 1 to be observed in the figure, and epi-illumination is performed from below the objective lens 2. . The light flux from the light source 11 for epi-illumination is condensed by the collector lens 12 to form an image, and then the lens 15
And enters the dichroic mirror DM via the excitation filter EX1.

A field stop FS1 is arranged at an image forming position between the collector lens 12 and the lens 15.
Further, the dichroic mirror DM is arranged in the relay lens system 3 (in the parallel optical path) of the observation optical system of the microscope. The epi-illumination light flux reflected upward in the figure by the dichroic mirror DM illuminates the observation field region of the sample 1 via the objective lens 2. The sample 1 and the field stop FS1 are at a conjugate position, and the observation field area is defined depending on the shape and size of the opening of the field stop FS1.

In this embodiment, the B excitation method is used, and the excitation filter EX1 has a characteristic of transmitting light having a wavelength of 430 to 480 nm, that is, excitation light. On the other hand, the dichroic mirror DM reflects light having a wavelength of 330 to 500 nm, and the wavelength generated in the sample illuminated by epi-illumination is 500.
It has the property of transmitting only light of nm or more. In this way, the light source 11, the collector lens 12, the field stop FS1,
The lens 15, the excitation filter EX1, the dichroic mirror DM, and the objective lens 2 constitute an excitation light irradiation optical system of the epi-illumination fluorescent lighting device.

On the other hand, the light source 16 for irradiating the destructive light for destroying the caged compound is, for example, a xenon light source connected to a high voltage power source (not shown) and emits flash light. Light from the xenon light source 16 is collected by the collector lens 17
The light is focused by and is focused on the input end 18a of the optical fiber 18. The light propagating through the fiber 18 reaches the exit end 18b and is emitted as a secondary light source. The divergent light from the exit end 18b is the objective lens 2 arranged above the sample 1 in the figure.
Illuminate the specimen 1 through 0 to destroy the caged compound contained in the specimen.

A field stop FS2 is arranged near the exit end 18b of the fiber 18 at a position conjugate with the sample 1 with respect to the objective lens 20. The field stop FS2 can narrow the opening and can be two-dimensionally moved in a plane orthogonal to the optical axis. In this way, the irradiation area of the destructive light on the sample 1 is defined by the field stop FS2.

An excitation filter EX2 is arranged between the field stop FS2 and the objective lens 20. The excitation filter EX2 has a characteristic of transmitting light having a wavelength of 330 to 380 nm as ultraviolet light for destroying the caged compound. As described above, the xenon light source 16, the collector lens 17, the fiber 18, the field stop FS2, the excitation filter EX2, and the objective lens 20 configure the destructive light irradiation optical system of the epi-illumination device.

In this way, the wavelength 5 generated in the sample irradiated with the excitation light from the light source 11 or the destructive light from the light source 16
The fluorescence of 00 nm or more is condensed by the objective lens 2 and then is collected by the dichroic mirror DM in the relay lens system 3.
Through. Here, after the light harmful to the observation is removed through the absorption filter BA which transmits the light having the wavelength of 520 nm or more, the fluorescence from the sample enters the mirror 4.
The light reflected by the mirror 4 obliquely upward and leftward in the drawing forms an enlarged image 5.

The light from the magnified image 5 is re-imaged through the relay lens system 6 and the prism 7 to form an image 8.
The image 8 is observed by the naked eye 10 of the observer through the eyepiece 9. In this way, the objective lens 2, the relay lens system 3, the mirror 4, the relay lens system 6, the prism 7 and the eyepiece lens 9 constitute the observation optical system of the microscope.

In this embodiment, the destructive light for destructing the caged compound is transmitted through the field stop FS2 to the objective lens 20.
Is focused on the sample 1. Therefore, it is possible to irradiate only a partial region of the observation visual field region of the sample 1 to which the excitation light for epi-illumination is irradiated with the destructive light and observe the reaction of some cells of the sample. Further, by appropriately narrowing the field stop FS2, it is possible to efficiently irradiate a desired small area with the destructive light having a desired intensity within the observation field area of the sample 1. As a result, it is possible to illuminate a stronger excitation light in a smaller area.

Furthermore, by appropriately moving the field stop FS2 two-dimensionally in the plane orthogonal to the optical axis, it is possible to selectively irradiate only a specified region of a part of the cells of the sample 1 with the excitation light. In other words, it is possible to perform fluorescence observation over the entire observation visual field region while sequentially irradiating the arbitrary region of the observation visual field region with the destructive light. Further, the exit end 18b of the fiber 18, which is the secondary light source that supplies the excitation light, is two-dimensionally moved together with the field stop FS2, so that the specimen 1 is irradiated with the destructive light over the range of the diameter of the fiber 18 or more. It will be possible. Furthermore, the objective lens 2
By moving 0 along the optical axis, it is possible to correct the focus shift due to chromatic aberration and irradiate the destructive light on a very small area with good imaging performance.

FIG. 2 is a diagram showing the structure of the epi-illumination device for a microscope according to the second embodiment of the present invention. In FIG. 2, the observation optical system of the microscope is omitted, and the configuration of the epi-illumination illuminator is mainly shown. The device of the second embodiment has a similar configuration to the device of the first embodiment and has the same operation and effect, but the light for destroying the caged compound is guided to the sample through a part of the optical path of the observation optical system of the microscope. The difference is the difference from the first embodiment. That is, the second embodiment is largely different only in that the destructive light and the excitation light are partially condensed on the sample by the common objective lens via the common optical path, and thus the configuration and operation similar to those of the first embodiment are performed. The overlapping description of is omitted.
In the apparatus of FIG. 2, elements having the same functions as those of the components shown in FIG. 1 are designated by the same reference numerals.

In the excitation light irradiation optical system of the epi-illumination fluorescent lighting device of FIG. 2, the light flux from the light source 11 is condensed by the collector lens 12 and once focused, and then the collimator lens 13 is formed.
Is converted into a parallel light flux by. This parallel light flux enters the dichroic mirror DM2 via the excitation filter EX1.

A field stop FS3 is arranged at an image forming position between the collector lens 12 and the collimator lens 13. The parallel light beam for epi-illumination reflected by the dichroic mirror DM2 on the left side in the drawing is imaged by the lens 14, becomes a parallel light beam by the collimator lens 15, and enters the dichroic mirror DM1. Here, a field stop FS1 is arranged at an image forming position between the lens 14 and the collimator lens 15. The dichroic mirror DM1 is arranged in the relay lens system 3 (in the parallel optical path) of the observation optical system of the microscope.

The parallel light flux reflected upward in the figure by the dichroic mirror DM1 illuminates the observation field area of the sample 1 via the objective lens 2. As described above, the sample 1 and the field stop FS1 and the field stop FS3 are at conjugate positions, and the observation field area is the field stop FS1 and the field stop FS.
3 is defined depending on the shape and size of the opening.

The B excitation method is also used in the second embodiment, and the excitation filter EX1 has a characteristic of transmitting light having a wavelength of 430 to 480 nm. On the other hand, the dichroic mirror DM1 has a characteristic of reflecting light having a wavelength of 330 to 500 nm and transmitting only light having a wavelength of 500 nm or more generated in a sample illuminated by epi-illumination. Further, the dichroic mirror DM2 has a property of reflecting light having a wavelength of 400 to 660 nm and transmitting light having a wavelength of 280 to 390 nm. Thus, the dichroic mirror DM2 reflects the excitation light (430 to 480 nm) from the light source 11 and transmits the destructive light (330 to 380 nm) from the light source 16 described later.

On the other hand, in the destructive light irradiation optical system of the epi-illumination device of FIG. 2, the light from the xenon light source 16 is condensed by the collector lens 17, and the input end 1 of the optical fiber 18 is collected.
Form an image on 8a. The light propagating through the fiber 18 is the exit end 1
8b, it becomes a secondary light source and is emitted. Ejection end 18b
The divergent light from the light enters the above-mentioned dichroic mirror DM2 via the collimator lens 19 movable along the optical axis and the excitation filter EX2. The light transmitted through the dichroic mirror DM2 illuminates the sample 1 through the lens 14, the collimator lens 15, the dichroic mirror DM1 and the objective lens 2 to destroy the caged compound, similarly to the parallel light flux for epi-illumination described above. .

A field stop FS2 is arranged near the exit end 18b of the fiber 18 at a position conjugate with the sample 1. The field stop FS2 can narrow the opening and can be two-dimensionally moved in a plane orthogonal to the optical axis. Further, the excitation filter EX2 has a wavelength of 330 to 330 as ultraviolet light for destroying the caged compound.
It has a characteristic of transmitting light of 380 nm. Also in the present embodiment, by moving the collimator lens 19 along the optical axis, it is possible to correct the focus shift due to chromatic aberration and irradiate the destructive light on a very small area with good imaging performance.

In each of the above-mentioned embodiments, the present invention has been described by taking an inverted microscope as an example, but the present invention can also be applied to a general upright microscope. Further, in each of the above-described embodiments, the configuration according to the B excitation method is shown as the epifluorescence method, but other suitable excitation method may be used. Further, in each of the above-mentioned embodiments, the secondary light source of the destructive light is formed via the optical fiber, but the field stop may be arranged directly in the vicinity of the light source for the destructive light without using the optical fiber. Is clear.

[0030]

As described above, according to the present invention, since the destructive light is focused on the sample by the condenser lens means through the field stop, the reaction of a part of the cells of the sample can be observed. . Further, by making it possible to narrow down the field stop, it is possible to irradiate a region of a desired size with the destructive light of a desired intensity. Furthermore, by making the field stop movable two-dimensionally in a plane perpendicular to the optical axis, fluorescence can be observed over the entire observation visual field area while sequentially irradiating destructive light to any area of the observation visual field area. It can be carried out.

[Brief description of drawings]

FIG. 1 is a diagram showing the configuration of an epi-illumination device for a microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing the configuration of an epi-illumination device for a microscope according to a second embodiment of the present invention.

[Explanation of symbols]

 1 sample 2 objective lens 3 relay lens system 4 mirrors 5 and 8 image plane 7 prisms 11 and 16 light source 18 fiber EX excitation filter BA absorption filter DM dichroic mirror FS field stop

Claims (7)

[Claims] 1. An excitation light irradiation optical system for irradiating a specimen with excitation light for epi-illumination, and a destructive light for irradiating the specimen with destructive light for destroying a caged compound contained in the specimen. In a microscope epi-illumination fluorescent illumination device including an irradiation optical system, the destructive light irradiation optical system condenses a light source for emitting the destructive light and light from the light source in an observation visual field region of the sample. And a field stop for defining an irradiation area of the destructive light on the sample, which is positioned between the light source and the light collecting means. Fluorescent lighting device. 2. The epi-illumination device as claimed in claim 1, wherein the field diaphragm can be narrowed down. 3. The epi-illumination illuminator according to claim 2, wherein the field stop is movable in a plane perpendicular to the optical axis. 4. The epi-fluorescent light as claimed in claim 3, wherein the light source is arranged in the vicinity of the field stop and is movable integrally with the field stop in a plane perpendicular to the optical axis. Lighting equipment. 5. A second light collecting unit for collecting light from the light source.
And a light guide means for propagating the incident light to the exit end, which has an entrance end at a position where it is focused by the second condensing lens means. 4. The exit end is arranged in the vicinity of the field stop, and the light from the exit end is condensed into the observation field area of the sample via the field stop. The epi-fluorescent illumination device according to item 1. 6. The epi-illumination device as claimed in claim 5, wherein the exit end of the light guide means is movable integrally with the field stop in a plane perpendicular to the optical axis. 7. At least one lens of the lenses forming the condenser lens means is a movable lens movable along an optical axis, and the movable lens is moved along the optical axis to move the field stop. The epi-fluorescent illumination device according to claim 1, wherein focusing is performed on the sample.