JPH09329748A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a good quality observation image and to stably perform experiment and observation using cells by forming the sectional area of an optical fiber bundle to be larger than a region irradiated with a light transmitted through Nipkow's disk. SOLUTION: The end surfaces 60a and 60b of an optical fiber bundle 60 have sectional areas larger than a region 122 irradiated with a light transmitted through a pinhole 41 following the rotation of Nipkow's disk 40. A reflected light from a sample 20 is passed on the optical path of a direction reverse to the irradiation light, in sequence through an objective lens 73, a mirror 72, a lens 71 and the optical fiber bundle 60, and then the reflected light is focused in the pinhole 41 of the Nipkow's disk 40. The reflected light passed through the pinhole 41 is reflected by a half mirror 13 and guided to an eyepiece 31. At this time, the Nipkow's disk 40 is rotated at a high speed by a motor 50 and, since the reflected light obtained by luster-scanning the sample 20 is left as a remaining image on a retina, the sample 20 is observed as an image by eyes.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope, and more particularly to a confocal microscope which uses a Nipkow disk for optical scanning.

[0002]

2. Description of the Related Art FIG. 3 is a block diagram of a conventional confocal microscope using a Nipkow disk.

The confocal microscope 100 includes a light source 101 and a Nipkow disk 10 rotated by a motor 102.
3 and light emitted from the light source 101 through the condenser lens 104 and the half mirror 105.
3, the pinholes 103a, 103b, 10 which are the translucent parts.
Objective lens 108 for focusing light on the sample 107 via 3c
And an eyepiece 110 for observing the reflected light CL moving in the range of arrow p with the rotation of the Nipkow disk 103 with the naked eye 109.

FIG. 4 is a plan view of the Nipkow disk.

A plurality of pinholes 103a, 103b, 103c formed by etching are spirally arranged at a predetermined interval on a disk formed by vapor-depositing metal on flat glass.

FIG. 5 is a diagram for explaining scanning by the Nipkow disk, and the light source 101 with the Nipkow disk 103 interposed therebetween.
122 on the screen 120 located on the opposite side of the
A case of scanning (corresponding to the sample 107 in FIG. 3) will be described.

The light emitted from the light source 101 is reflected by the lens 1
The light beam is made into parallel rays by 04, and the area A surrounded by the dotted line on the upper surface of the Nipkow disk 103 is irradiated. Of this irradiation light, only the light that has passed through the pinhole 103a is focused on the area 122 on the screen 120 by the objective lens 108.

The spot 121a formed by the light passing through the pinhole 103a moves in the area 122 from the bottom to the top of FIG. 5 while drawing an arc-shaped locus a as the Nipkow disk 103 rotates clockwise.

When the Nipkow disk 103 further rotates and the spot 121a leaves the area 122, the spot 121b formed by the light passing through the next pinhole 103b.
, Like the spot 121a, moves from top to bottom while drawing an arc-shaped trajectory b. At this time, since the pinhole 103b is formed on a smaller radius than the pinhole 103a, the locus b of the spot 121b is also laterally displaced by that amount as shown in the figure.

When the Nipkow disk 103 further rotates,
The movement of the spot 121c formed by the light that has passed through the next pinhole 103c forms a locus c that is further laterally displaced from the locus b of the spot 121b.

By rotating the Nipkow disk 103 in this manner, the spot light that passes through the Nipkow disk 103 and is applied to the sample can be raster-scanned on the area 122.

[0012]

However, in scanning by the Nipkow disk 103, since the Nipkow disk 103 is rotated at a high speed by using the motor 102, vibration is inevitably generated. On the other hand, in the confocal microscope 100 using the conventional Nipkow disk 103, the motor 102 and the objective lens 108 are integrated and housed in one housing 200 (see FIG. 3). Therefore, the vibration is transmitted to the objective lens 108 and the sample 107 through the housing 200 and the like, and the converging position of the irradiation light on the sample 107 is deviated, so that the obtained observation image has poor image quality. was there. Further, when performing a biological experiment by fixing or manipulating the cells, there is a problem that the cells move due to vibration and stable experiments and observations cannot be performed.

The present invention has been made in view of the above circumstances, and its problem is a confocal microscope capable of obtaining an observation image with high image quality and stably performing experiments and observations using cells. Is to provide.

[0014]

In order to solve the above-mentioned problems, the confocal microscope according to the invention of claim 1 is a Nipkow disk in which a light source and a plurality of light-transmitting parts are spirally arranged on the disk at predetermined intervals. In a confocal microscope comprising: a microscope body that collects light emitted from the light source onto a sample through a light transmitting portion of the Nipkow disk, and transmits light passing through the light transmitting portion of the Nipkow disk to the microscope body. And a cross-sectional area of the optical fiber bundle is larger than an area irradiated by the light transmitted through the transparent portion of the rotating Nipkow disk.

The light emitted from the light source passes through the transparent portion of the Nipkow disk, is further transmitted to the microscope main body through the optical fiber bundle, and is irradiated onto the sample. The reflected light from the sample travels back through the optical fiber bundle, and only the light from the focal plane of the sample passes through the light transmitting portion at a position conjugate with the focal plane of the sample. Since the light moves two-dimensionally in a part of one end face of the optical fiber bundle as the Nipkow disk rotates, the light is two-dimensionally irradiated in the observation region of the sample. At this time, since the Nipkow disk and the microscope body are connected via the optical fiber bundle, it is possible to prevent the irradiation position of the irradiation light on the sample from being shaken by the vibration of the Nipkow disk, and it is possible to observe the cells and operate the cells. It is possible to prevent the cells from moving during this.

According to a second aspect of the present invention, in the confocal microscope according to the first aspect, the optical fiber bundle receives the light emitted from the sample and transmits the light to the Nipkow disk, and the confocal microscope includes the optical fiber bundle and It is characterized by further comprising light separating means for separating the light from the sample obtained through the Nipkow disk from the light from the light source to form a sample image. The optical fiber bundle transmits the light from the light source to the microscope body and the light from the sample obtained from the microscope body to the Nipkow disk. That is, it is possible to transmit the illumination light and the sample image.

[0017]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of a reflection type confocal microscope according to the first embodiment of the present invention.

The confocal microscope 1 includes a light source 11 for emitting illumination light, a lens 12 for collimating the light emitted from the light source 11 and the illumination light, but a sample 20.
A half mirror 13 for reflecting the reflected light from the eye, an eyepiece lens 31 for condensing the light reflected by the half mirror 13 on the eye 30, and a plurality of pinholes (light transmitting parts) 41 for passing the light. The formed Nipkow disk 40, the motor 50 for rotating the Nipkow disk 40, the lens 14, the optical fiber bundle 60 having the one end surface 60a arranged at the focal plane of the lens 14, and the other end surface 60b of the optical fiber bundle 60 A lens 71 serving as a focal plane, a mirror 72 that changes the directions of the illumination light and the reflected light from the sample 20, and an objective lens 73 that focuses the illumination light on the surface of the sample 20.

In this embodiment, the light source 11 and the lens 1
2, the half mirror 13, the eyepiece lens 31, the Nipkow disk 40, the motor 50, and the lens 14 are housed in the housing 10, and the microscope main body including the lens 71, the mirror 72, and the objective lens 73 is housed. The housing 10 and the housing 70 are arranged apart from each other, and the housings 10 and 70 are connected by an optical fiber bundle 60 to transmit illumination light and reflected light.

It is not necessary to use laser light as the light source 11, and an arc lamp such as a Xe lamp or a Hg lamp can be used.

As the optical fiber 60, an image transmission optical fiber having a diameter of 20 to 30 mm, which is used in a fiberscope or the like, is used. This is an integrated optical fiber in which several thousand to tens of thousands of optical fibers each corresponding to one pixel are collected. The end faces 60a, 6 of the optical fiber bundle 60
0b has a larger cross-sectional area than the area 122 illuminated by the illumination light passing through the pinhole 41 as the Nipkow disk 40 rotates.

Next, the operation of the confocal microscope 1 having the above configuration will be described.

The illumination light emitted from the light source 11 is used by the lens 1
The light is collimated by 2 and transmitted through the half mirror 13, and then the upper surface of the Nipkow disk 40 is irradiated. Of this irradiation light, only the illumination light that has passed through the pinhole 41 is condensed by the lens 14 on the one end surface 60a of the optical fiber bundle 60.

The condensed illumination light propagates in the optical fiber bundle 60 and is emitted from the other end surface 60b. The illumination light is condensed by the lens 71, reflected by the mirror 72, and then condensed by the objective lens 73 on the focal plane on the sample 20.

The reflected light from the sample 20 is the objective lens 7
3, the mirror 72, the lens 71, and the optical fiber bundle 60 follow the optical path in the opposite direction to the irradiation light, and then the lens 14 focuses the pinhole 41 of the Nipkow disk 40. At this time, only the light reflected by the focal plane on the sample 20 passes through the pinhole 41 due to the confocal effect, and the scattered light and the like from the surface of the sample 20 are not focused on the pinhole 41 and are therefore removed.

The reflected light that has passed through the pinhole 41 is reflected by the half mirror 13 and guided to the eyepiece lens 31.

At this time, the Nipkow disk 40 is moved to the motor 5
It is rotated at high speed by 0 (several thousands of revolutions per minute)
The reflected light obtained by raster scanning (two-dimensional scanning) above can be left on the retina as an afterimage, so that the sample 20 can be visually observed as an image.

If a TV camera (not shown) is arranged instead of the eyepiece lens 31, the sample 20 can be observed through the TV monitor.

FIG. 2 is a block diagram of an epi-fluorescence type confocal microscope according to the second embodiment of the present invention. Portions common to the first embodiment described above are denoted by the same reference numerals, and description thereof is omitted.

In the second embodiment, the dichroic mirror 113 is arranged instead of the half mirror 13, and the dichroic mirror 113 and the lens 12 are used.
Between the dichroic mirror 113 and the eyepiece 31
The difference from the first embodiment is that the excitation filter 15 and the absorption filter 32, which correspond to the fluorescence to be observed and the excitation light, are respectively disposed between and.

Next, the operation of the epi-fluorescence confocal microscope 5 having the above construction will be described.

The light emitted from the light source 11 such as an arc lamp is collimated by the lens 12, and the excitation filter 1
Light other than the excitation wavelength is removed by 5, and after passing through the dichroic mirror 113, the upper surface of the Nipkow disk 40 is irradiated. Of this excitation light, only the excitation light that has passed through the pinhole 41 is passed through the lens 14 to the optical fiber bundle 6
The light is focused on one end surface 60a of 0.

The condensed excitation light propagates in the optical fiber bundle 60 and is emitted from the other end face 60b. The excitation light is collected by the lens 71, reflected by the mirror 72, and then collected by the objective lens 73 on the focal plane on the cell 20 which is dyed with a fluorescent dye in advance. Therefore, sample 20
The fluorescent substance previously injected therein is excited to generate fluorescence.

The fluorescence follows the objective lens 73, the mirror 72, the lens 71, the optical fiber bundle 60, and the optical path in the opposite direction of the excitation light, and then the lens 14 causes the Nipkow disk 4 to pass.
The zero pinhole 41 is focused. At this time, only the fluorescence condensed on the focal plane on the sample 20 passes through the pinhole 41 due to the confocal effect.

The fluorescence passing through the pinhole 41 is reflected by the dichroic mirror 113, and the eyepiece 3
Guided to 1.

At this time, the Nipkow disk 40 is connected to the motor 5
The fluorescence obtained by raster scanning (two-dimensional scanning) on the sample 20 at a high speed by 0 can be left on the retina as an afterimage, so that the sample 20 can be observed as an image with the naked eye 30.

As in the case of the first embodiment, it is possible to observe the sample through the TV monitor by disposing a TV camera (not shown) instead of the eyepiece lens 30.

[0039]

As described above, according to the confocal microscope of the first aspect of the present invention, since the Nipkow disk and the microscope main body are connected via the optical fiber, the vibration of the Nipkow disk causes the vibration on the sample. It is possible to prevent the irradiation position of the irradiation light from moving, to prevent the cells from moving during cell observation, to prevent deterioration of the quality of the observation image, and to perform experiments and observations using cells. It can be performed stably.

According to the confocal microscope of the second aspect, the optical fiber bundle transmits the light from the light source to the microscope body and the light from the sample obtained from the microscope body to the Nipkow disk. That is, it is possible to transmit the illumination light and the sample image. Therefore, the confocal microscope can have a simple structure and can obtain a stable sample image.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a reflection-type confocal microscope according to a first embodiment of the present invention.

FIG. 2 is a configuration diagram of an epi-fluorescence confocal microscope according to a second embodiment of the present invention.

FIG. 3 is a configuration diagram of a conventional confocal microscope using a Nipkow disk.

FIG. 4 is a plan view of a Nipkow disk.

FIG. 5 is a diagram illustrating scanning by a Nipkow disk.

[Explanation of symbols]

 1 Confocal Microscope 11 Light Source 20 Sample 40 Nipkow Disc 41 Pinhole (Transparent Portion) 60 Optical Fiber 122 Area

Claims (2)

[Claims] 1. A light source, a Nipkow disk in which a plurality of light-transmitting portions are spirally arranged on a disk at predetermined intervals, and light emitted from the light source is condensed on a sample through a light-transmitting portion of the Nipkow disk. In a confocal microscope including a microscope body to be made to have an optical fiber bundle for transmitting light passing through a transparent portion of the Nipkow disk to the microscope body, the cross-sectional area of the optical fiber bundle is the rotating Nipkow disk. The confocal microscope is characterized in that it is larger than the area illuminated by the light transmitted through the transparent portion of the. 2. The optical fiber bundle receives the light emitted from the sample and transmits the light to the Nipkow disk, and the confocal microscope transmits the light from the sample obtained through the optical fiber bundle and the Nipkow disk to the light source. The confocal microscope according to claim 1, further comprising: a light separating unit that separates the light from the sample to form a sample image.