JP2000097858A - Scanning type confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire a multi-chromatic fluorescent image requiring no optical regulation of high precision. SOLUTION: This microscope has a light source to emit excited light of at least one single wave length, an optical system for converging the excited light on a sample to irradiate the sample, a scanning means for scanning two- dimensionally a condensed point of the excited light, a confocal optical system 40, an optical system for introducing fluorescence from the sample to it, a spectroscopic means 41, and a fluorescence detecting optical system. The confocal optical system 40 includes a lens for converging the fluorescence, a confocal diaphragm having a positional relation conjugated all the time with the condensed point of the excited light inside the sample, and a lens for collimating the transmitted fluorescence. The spectroscopic means 41 comprises preferably a prism. The fluorescence detecting optical system has a diffusion mirror 42 for diffusing lights separated into it spectral components, plural converging lenses 46, 50, 54 for converging the light reflected by the mirror 42, and detectors 47, 51, 55 paired with the lenses 46, 50, 54.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning confocal microscope used for medical / biological research and various industrial measurements, and particularly for simultaneously detecting a plurality of fluorescences.

[0002]

2. Description of the Related Art At present, a sample is irradiated with light of a specific wavelength,
Scanning confocal microscopes for observing fluorescence from a sample excited by the wavelength are well known. In biomedical research in particular, it has become an important research tool in observing cell morphology and studying physiological phenomena.In recent years, it has been widely used. The development of reagents is being actively pursued.

[0003] Depending on the purpose of the research, it has become increasingly important to obtain not only single-light fluorescence observation but also images with good S / N of two-color and three-color multi-stained samples. . In a scanning microscope that has become widespread at present, fluorescence detection during multiple staining is performed using a plurality of wavelength-dependent filters. In this method, it is necessary to prepare a large number of filters according to the observation wavelength, and it is necessary to prepare filters one after another in accordance with the spectral characteristics of a newly developed fluorescent reagent.

As a method of solving such inconvenience, a method of observing fluorescence using optical spectrum without using a filter having a wavelength dependency has been proposed.
Such examples are disclosed in Japanese Patent No. 43737 and Japanese Patent Publication No. 9-502269.

FIG. 4 shows the configuration of the apparatus disclosed in Japanese Patent Application Laid-Open No. 8-43939. The laser light emitted from the laser light sources 1 and 20 passes through the beam expanders 2 and 2, the dichroic mirror 22, the laser line filter 3, and the dichroic mirror 4, and then passes through the XY scanning system 5, the pupil projection lens 6, and the microscope 7. Are sequentially irradiated to the sample 19. The fluorescence from the sample 19 passes through the microscope 7, the pupil projection lens 6, the XY scanning system 5, and then passes through the dichroic mirror 4.
The laser beam is separated from the laser beam by the laser beam and guided to the confocal optical system 8. The fluorescence that has passed through the pinhole of the confocal optical system 8 is split by the grating 9 by being deflected at an angle corresponding to the wavelength. The separated fluorescence of each wavelength passes through the slits 10, 11, and 12 and the condenser lenses 16, 17, and 18, respectively, and passes through the photodetectors 13, 14, and 15, respectively.
Incident on. The slit width of each of the slits 10, 11, 12 is suitably changed according to the fluorescence wavelength to be detected.

FIG. 5 partially shows an apparatus disclosed in Japanese Patent Publication No. 9-502269, and shows an optical system for spectrally detecting and detecting fluorescence. This optical system includes a prism 27 for splitting the fluorescent light 24 by deflecting the fluorescent light 24 at an angle corresponding to the wavelength, and selectively transmitting only the light 29 in the first wavelength range among the split light, and reflecting the other light. First diaphragm 28
A first detector 31 that detects light 29 in a first wavelength range that has passed through the first stop 28; and a light 3 reflected by the first stop 28.
0, selectively pass only the light 34 in the second wavelength range,
Other than that, it has a second stop 33 that reflects light, and a second detector 32 that detects light 34 in a second wavelength range that has passed through the second stop 33. The relative angle and position of the diaphragms 28 and 33 can be changed. Although not shown in this figure,
It further includes a third stop that selectively allows only light in the third wavelength range among light reflected by the second stop 33, and a third detector that detects light of the third wavelength that has passed through the third stop. Examples are also disclosed.

[0007]

As is well known, whether or not a grating can be split according to the polarization direction of light is determined. Light split by the grating is limited to light having a specific polarization direction. Generally, the fluorescence from the sample is randomly polarized,
Light split by the grating is limited to a small portion of the fluorescence from the sample. Accordingly, the above-mentioned Japanese Patent Application Laid-Open No.
As represented by No. 3739, an apparatus using a grating for spectral separation has poor spectral efficiency, and the obtained image tends to have insufficient brightness.

[0008] Further, the spread angle of the light split by the prism is small, and even if the light from UV light to IR light is split,
The obtained spread angle is at most about 5 °. For this reason, as typified by the above-mentioned Japanese Patent Application Laid-Open No. 8-43739, in an apparatus using a prism for spectral separation, various optical elements for detecting fluorescence include, for example, diaphragms 28 and 33 and detectors 31 and 32.
Are required to be arranged with very high positional accuracy. This makes it difficult to perform optical adjustment at the time of assembling, not only lowering the productivity but also lowering the measurement accuracy.

Furthermore, in the apparatus disclosed in Japanese Patent Application Laid-Open No. 8-43939, the arrangement of a plurality of detectors is not the same, and the distance from the confocal pinhole to each detector is different. This is the reliability of the measurement results obtained based on each detector,
In particular, it becomes a factor that lowers the reliability of mutual evaluation between them.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a scanning confocal microscope capable of acquiring a fluorescent image by multiple staining, which does not require high-precision optical adjustment. is there.

[0011]

A scanning confocal microscope according to the present invention comprises: a light source for emitting at least one excitation light of a single wavelength; an irradiation means for condensing and irradiating the sample with excitation light; Scanning means for two-dimensionally scanning the light converging point;
It has fluorescence detection means for detecting fluorescence generated in the sample, and a confocal stop arranged at a position conjugate with the converging point of the excitation light, and the fluorescence detection means is configured to detect the fluorescence from the sample passing through the confocal stop. Spectroscopic means for deflecting the fluorescence of the light according to the wavelength and separating the light,
A light diffusing means for diffusing the dispersed light to increase its opening angle, a plurality of condensing means for condensing light in different wavelength ranges included in the diffused light, and condensing by each condensing means And a plurality of light detection means for detecting the respective light beams.

[0012] In a preferred example, the fluorescence detecting means is:
The light-emitting device further includes a light-shielding plate that blocks light in a wavelength range corresponding to the excitation light from the light source included in the diffused light. Further, in another preferred example, the fluorescence detecting means further includes a movable light shielding plate capable of selectively restricting light incident on the light collecting means.

[0013]

Embodiments of the present invention will be described below with reference to the drawings. [First Embodiment] FIG. 1 shows a part of a scanning confocal microscope according to a first embodiment.
2 shows a confocal optical system, a spectral unit, and a fluorescence detection optical system.

Although not shown in FIG. 1, the scanning confocal microscope includes a light source capable of emitting at least one single-wavelength excitation light, and an optical source for condensing and irradiating the sample with the excitation light. The system includes a system, a scanning unit that two-dimensionally scans the focal point of the excitation light, and an optical system that guides fluorescence from the sample generated by the excitation with the excitation light to the confocal optical system 40.

The light source may be constituted by a single light source capable of emitting light of a plurality of wavelengths, or may be constituted by a plurality of light sources capable of emitting light of a single wavelength. Good.

These structures are the same as those of a normal scanning confocal microscope, and for example, the structure shown in FIG. 4 can be applied as it is. As shown in FIG. 1, the scanning confocal microscope further includes a confocal optical system 40, a spectral unit 41, and a fluorescence detecting optical system.

The confocal optical system 40 includes a lens for condensing incident fluorescent light, a confocal stop which is always in a conjugate positional relationship with the converging point of the excitation light, and a lens for collimating the fluorescent light passing through the confocal stop. And

Since the confocal stop is always in a conjugate positional relationship with the condensing point of the excitation light, only the fluorescent light generated at the condensing point and its very near position can pass through the confocal optical system 40. The parallel light flux 43 of the fluorescent light that has passed through the confocal optical system 40 is incident on the spectroscopic means 41 and is separated at an angle corresponding to the wavelength.

Although a prism or a grating can be applied to the spectroscopic means 41, a prism having a small loss is preferably used. For this reason, in the present embodiment, a prism is used for the spectral unit. The prism 41 preferably has a light flux 43 to reduce reflection loss on the incident surface.
Are arranged to enter at a Brewster angle.

The fluorescence detecting optical system includes a diffusing mirror 42 for diffusing the dispersed light, a plurality of condensing lenses 46, 50, and 54 for condensing the light reflected by the light, and a detector paired with these. 47, 51, and 55. The diffusing mirror 42 is constituted by, for example, a part or all of a reflecting surface formed of a cylindrical surface, or a part or all of a reflecting surface formed of a spherical surface, but is not limited thereto. The diffusing mirror 42
Any optical element having a function of widely diffusing the light dispersed by the prism 41 in a one-dimensional direction or a two-dimensional direction can be applied. The detectors 47, 51, 55 preferably use a photomultiplier tube (PMT) in consideration of detection sensitivity and the like.

The light split by the prism 41 is diffused by a diffusing mirror 42.
Widely spread by. Short-wavelength light, for example, blue light is highly polarized by the prism 41, and long-wavelength light, for example, red light, is lightly polarized. Accordingly, in the light diffused by the diffusion mirror 42, in FIG. 1, short-wavelength light, for example, blue light is arranged on the left side, and long-wavelength light, for example, red light is arranged on the right side.

The split light includes fluorescence from the sample and reflected light of laser light. FIG. 1 shows an example in which the laser light having three wavelengths is excited by the laser light.
4 and the fluorescence 45 excited thereby, for example FITC,
A laser beam 48 having a wavelength of 543 nm and fluorescence 49 excited thereby, such as Rhodamine, a laser beam 52 having a wavelength of 633 nm, and fluorescence 53 excited thereby, such as Cy5
It is.

The condenser lenses 46, 50 and 54 are arranged on the optical paths of the fluorescent light 45, 49 and 53, respectively.
Photoelectron image tubes 47, 51, and 55 are disposed at the light condensing positions of the condenser lenses 46, 50, and 54, respectively.
The size and position of the condenser lenses 46, 50, 54 are selected such that the laser beams 44, 48, 52 do not enter while preferably receiving a large amount of fluorescence 45, 49, 53, respectively. The thickness of the light beam 43 and the prism 41
The distance between the light source and the diffusion mirror 42 is determined so that light of each wavelength is sufficiently separated.

According to the present embodiment, since the light split by the prism 41 is diffused by the diffusion mirror 42, the spread angle is enlarged. Therefore, the condenser lenses 46 and 5
The positional accuracy required for the detectors 0, 54 and the detectors 47, 51, 55 is lower than the configuration without the diffusion mirror 42. This facilitates assembling and improves productivity. This in turn helps to reduce the manufacturing costs of the device.

The laser beams 44, 48, 52 and the fluorescent light 4
Since the separability of 5, 49, and 53 is improved, a high-quality image with a high S / N and little overlap between them can be obtained. Further, since the detectors 47, 51 and 55 are all equidistant from the pinhole of the confocal optical system 40, the images obtained through the respective detectors 47, 51 and 55 are homogeneous. This is preferable as a reliable information source when comparing and evaluating the respective images with each other.

[Second Embodiment] FIG. 2 shows a part of a scanning confocal microscope according to a second embodiment.
More specifically, a confocal optical system, a spectral unit, and a fluorescence detection optical system are shown. In the drawing, members equivalent to those already described in the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted to avoid duplication of description and will be omitted in the following description.

In this embodiment, the condenser lenses 46, 50,
A light shielding plate 56, 57, 58 for shielding unwanted laser light 44, 48, 52 is provided before 54. The other configuration is the same as that of the first embodiment.

According to this embodiment, the laser beams 44, 4
Since the light-shielding plates 56, 57, 58 for shielding the light beams 8, 52 are provided, the laser beams 44, 48, 52 are prevented from becoming stray light and overlapping the fluorescent light beams 45, 49, 53.
Images with good N can be obtained.

[Third Embodiment] FIG. 3 shows a part of a scanning confocal microscope according to a third embodiment.
More specifically, a confocal optical system, a spectral unit, and a fluorescence detection optical system are shown. In the drawing, members equivalent to those already described in the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted to avoid duplication of description and will be omitted in the following description.

In this embodiment, the condenser lenses 46, 50,
In front of 54, movable light shielding plates 60, 61, 62 capable of selectively restricting fluorescent light 45, 49, 53 incident thereon are provided. The other configuration is the same as that of the first embodiment.

According to the present embodiment, the movable light shielding plate 60,
The apertures of 61 and 62 are narrowed, and the laser beams 44 and 4 from the fluorescences 45, 49 and 53 incident on the detectors 47, 51 and 55
Eliminating the components that may cause overlap with the light shielding plates 8 and 52 obtains an image with a good S / N ratio.
The aperture of 0,61,62 is widened to increase the amount of fluorescence 45,4.
By making the light beams 9, 53 incident on the detectors 47, 51, 55, an image with high contrast can be obtained.

In the above-described embodiment, the configuration in which the sample is excited by the laser beams of three types of wavelengths has been described, but the number of types of the laser beams is not limited to this. The present invention is not limited to the above-described embodiments, but includes all implementations that do not depart from the gist of the invention.

Therefore, the following can be said about the scanning confocal microscope in the present embodiment. 1. A laser light source that scans at least one laser beam of a single wavelength and irradiates the sample with light, a detecting unit that detects light from the sample, an image forming unit that images light from the sample, and an image forming unit A confocal stop located at the focal position of the means;
At least one spectroscopic unit that divides the light that has passed through the confocal stop for each wavelength, a light diffusing unit for diffusing the split light, a light condensing unit that condenses the diffused light, A scanning confocal microscope, comprising: a light detecting means for detecting condensed light. 2. 2. The scanning confocal microscope according to claim 1, further comprising a light-shielding plate for blocking a wavelength corresponding to the laser light source included in the diffused light. 3. 2. The scanning confocal microscope according to claim 1, wherein the diffused light is selectively detected by a movable light shielding plate. 4. The light shielding plate according to claim 1, wherein a light shielding plate for blocking a wavelength corresponding to the laser light source from the diffused light and a movable light shielding plate for selectively detecting the separated light are provided. Scanning confocal microscope. 5. The scanning confocal microscope according to any one of claims 1 to 3, wherein the converging means and the detecting means are movable in accordance with a wavelength range to be detected.

[0034]

According to the present invention, there is provided a scanning confocal microscope which does not require high-precision optical adjustment and can obtain a fluorescent image by multiple staining. Since this scanning confocal microscope does not require high-precision optical adjustment, on the one hand, it is possible to manufacture it at low cost, and on the other hand, it is possible to acquire a fluorescent image with a good S / N. I do.

[Brief description of the drawings]

FIG. 1 shows a part of a scanning confocal microscope according to a first embodiment, and specifically shows a confocal optical system, a spectral unit, and a fluorescence detecting optical system.

FIG. 2 illustrates a part of a scanning confocal microscope according to a second embodiment, and specifically illustrates a confocal optical system, a spectral unit, and a fluorescence detection optical system.

FIG. 3 shows a part of a scanning confocal microscope according to a third embodiment, and specifically shows a confocal optical system, a spectral unit, and a fluorescence detecting optical system.

FIG. 4 shows a device configuration of a conventional example of a scanning confocal microscope capable of acquiring a fluorescent image by multiple staining.

FIG. 5 shows a spectroscopic section and a fluorescence detecting section in another conventional example of a scanning confocal microscope capable of acquiring a fluorescent image by multiple staining.

[Explanation of symbols]

 Reference Signs List 40 confocal optical system 40 41 spectral means 42 diffusing mirror 46, 50, 54 condensing lens 47, 51, 55 detector

Claims (3)

[Claims] 1. A light source that emits at least one excitation light of a single wavelength, an irradiation unit that collects and irradiates the excitation light onto a sample, and a scanning unit that two-dimensionally scans a focal point of the excitation light. And a confocal stop arranged at a position conjugate to the converging point of the excitation light. The fluorescence detecting means passes through the confocal stop. A spectroscopic means for deflecting the fluorescence from the sample according to the wavelength and dispersing the light, a light diffusing means for diffusing the dispersed light and increasing its opening angle, and a light of a different wavelength range included in the diffused light. A scanning confocal microscope comprising: a plurality of light condensing means for condensing light; and a plurality of light detecting means for detecting light condensed by each light condensing means. 2. The scanning confocal according to claim 1, wherein the fluorescence detecting means further comprises a light shielding plate for blocking light in a wavelength range corresponding to excitation light from a light source included in the diffused light. microscope. 3. The fluorescence detecting means further comprises a movable light shielding plate capable of selectively restricting light incident on the light collecting means.
The scanning confocal microscope according to claim 1.