JP2000227556A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the quantity of fluorescence which can be used for an observing action by enabling the fluorescence generated from a sample to be sufficiently utilized as a microscope constituted so that the observing action is executed by using the fluorescence. SOLUTION: In the vertical fluorescence microscope, an infinite focusing system objective lens 42 is placed in a space being opposite to an objective lens 22 by interposing the sample 21 and the focus thereof is aligned with that of the lens 22. Since light beams emitted from the lens 42 become the parallel light beams, the parallel light beams are reflected on a plane-surface mirror 44 and aligned with the optical path of the lens 22. Thus, an image can be formed by the lens 22 by putting the fluorescence directly emitted from the sample 21 and the fluorescence reflected on the mirror 44 together. As the result, the bright fluorescent image whose intensity of the fluorescence is increased can be observed through an eyepiece 25.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope for observing fluorescence emitted from a sample, and more particularly to an epifluorescence microscope, a confocal laser microscope, a multiphoton excitation laser microscope, and the like.

[0002]

BACKGROUND ART It is indispensable to understand the microstructure and the localization and movement of molecules when cells perform various functions in order to understand their mechanisms. There is a fluorescence microscope as a microscope used for observing this. In a fluorescence microscope, a fluorescent molecule that specifically binds to a specific molecule of interest in a specimen (this is called a fluorescent probe, for example, a fluorescent protein covalently bonded to an antibody of the protein of interest is used) And observe the distribution and movement of this molecule.

Now, a well-used epi-illumination fluorescence microscope will be described. Epi-illumination is an illumination method in which light from a light source is irradiated through an objective lens and fluorescence from a specimen is observed through the objective lens. The illumination light and the observation light pass through the same light from the objective lens. That is, the objective lens also serves as a condenser.

The configuration of an epi-fluorescence microscope having a configuration for realizing observation using this epi-illumination will be described with reference to FIG. In FIG. 1, in order to achieve a configuration for realizing observation by epi-illumination, an epi-fluorescence microscope is a dichroic microscope.
A mirror (DM) 24 is used. The dichroic mirror 24 is usually set in a small box called a dichroic cube together with an excitation filter and an absorption (barrier) filter. Irradiation light 32 of the light source is firstly cut off at an extra wavelength by an excitation filter 34, and passes only light having a wavelength that is absorbed by the fluorescent molecules used (see FIG. 2). This attenuates the background light that hinders observation.
This excitation light is reflected at a right angle by a dichroic mirror (DM) 24 inclined at 45 ° to the optical axis, and reaches the sample 21 through the objective lens 22. The fluorescence emitted from the sample 21 travels in the opposite direction to the excitation light and reaches the dichroic mirror (DM) 24 through the objective lens. As shown in FIG. 2, the dichroic mirror (DM) 24 has a property of reflecting light of a specific wavelength or less and transmitting light of a longer wavelength, so that the fluorescent light passes through this mirror. . After passing straight through, the absorption filter 36 cuts off wavelengths other than the fluorescence of interest to make the background as dark as possible. The above-mentioned epi-illumination fluorescence microscope uses information parallel to the stage surface in a region having a considerable thickness in the sample (light intensity distribution).
But do not give information in the optical axis direction.

One solution to this problem is a confocal laser-scanning microscope (CLSM).
Or a multiphoton excitation laser microscope (for example, a two-photon excitation laser microscope (TP-L
SM)). In each case, the sample surface is scanned with a laser to record the spatial distribution of the fluorescence on the focal plane, and a computer process is performed to reproduce the slice image.

A confocal laser microscope (CLSM) will be described with reference to FIGS. The confocal laser microscope uses single-photon excitation. The term “single-photon excitation” means that one fluorescent molecule is excited by absorbing one photon. This will be described in the case of single-photon excitation shown in FIG. FIG. 3 shows adiabatic potential curves 10 and 11, and the energy 12 of the photon from the laser turns the fluorescent molecule into an adiabatic potential curve 11 in an excited state. Then, when the state becomes the lower adiabatic potential curve 10 (arrow 14), the fluorescent light 15 is generated.

FIG. 4 shows a basic configuration of a confocal laser microscope using this fluorescence. A laser light 23 as a point light source is reflected by a dichroic mirror (DM) 24 that reflects the wavelength of the laser light on the sample 21, and an objective lens 22.
And irradiate. The fluorescence from the fluorescent molecule excited by the single photon by the laser light 23 passes through the wavelength of the fluorescent light.
It is stopped down by the lens 25 via the M24, passes through the confocal pinhole 27, and is received by the detector 26 composed of a photomultiplier tube or the like. In such a basic configuration, by scanning the laser beam 23 two-dimensionally with an XY scanning mirror (not shown), the fluorescent molecules in the optical path of the sample 21 irradiated with the laser beam 23 are excited. You. Of the fluorescence from the excited fluorescent molecules, only the fluorescence that has passed through the confocal pinhole 27 is received by the detector 26 and subjected to image processing by a computer, whereby a tomographic fluorescence image of the focal plane of the sample 21 is obtained. If the image of each tomographic plane is obtained by changing the focal plane and further image processing is performed, a three-dimensional image of the sample can be obtained.

In a confocal laser microscope (CLSM), the resolution in the optical axis direction is excellent. However, the laser penetrates through the sample and excites the fluorescent material in this region, so that a wide area of the fluorescent material is excited and many fluorescent molecules are discolored. As a result, the fluorescent molecules on the surface from which no image is acquired are also browned, so that the brown color of the fluorescent molecules in the sample becomes faster as a whole. In a measurement in which an image of the same focal plane is repeatedly acquired using free fluorescent molecules in cells, non-brown molecules are not sufficiently replenished from a region outside the focal plane where an image is not acquired, and browning is accelerated. Therefore, the brightness of the fluorescent image may be different between the beginning and the end of image acquisition. further,
Laser light is scattered when transmitting through a tissue having a complicated structure, and the sharpness of an image of a deep tissue (50 μm or more) is deteriorated or cannot be obtained. Further, in the case of an ultraviolet excitation fluorescent probe, there is a problem of cytotoxicity due to active oxygen generated by laser light or its irradiation, and there is a limit to an optical lens that can be used.

The multiphoton excitation laser microscope uses multiphoton excitation. The multiphoton excitation means that a single fluorescent molecule absorbs a plurality of photons almost simultaneously, thereby exciting the molecule with the sum of a plurality of photon energies. This will be described in the case of two-photon excitation shown in FIG.
FIG. 5 shows adiabatic potential curves 10 and 11 as in FIG. 3, and the energies 12 and 13 of the two photons result in an adiabatic potential curve 11 in which the fluorescent molecule is in an excited state. And the lower adiabatic potential curve 10
When the state (3) is reached (arrow 14), the fluorescent light 15 is generated.

As described above, the excitation wavelength of the two-photon excitation is twice as large as that of the ordinary single-photon excitation. The probability that a photon is absorbed by a fluorescent molecule is proportional to the photon density.
The probability of photon absorption is proportional to the square of the light intensity. Therefore,
Only the fluorescent molecules in the focal region in the sample are excited, and only the focal region emits fluorescence. In order for two photons to be absorbed by one molecule, the photon density must be increased. For this purpose, the energy of the excitation light is
It is temporally (<10 ⁻¹³ sec) by a laser and spatially concentrated by a lens. The pulse laser scans the tissue to obtain a two-dimensional image of the fluorescence, and obtains a three-dimensional image by changing the focal plane.

The two-photon excitation laser microscope (TP-LS)
FIG. 6 shows the basic configuration of M). A laser light 23 as a point light source is reflected on a sample 21 by a dichroic mirror (DM) 24 that reflects the wavelength of the laser light, and is focused by an objective lens 22 for irradiation. Fluorescence from a fluorescent molecule that is two-photon excited by the laser light 23 is narrowed down by a lens 25 via a DM 24 that passes the wavelength of the fluorescence, and
6 is received. In such a basic configuration, the laser light 23 is two-dimensionally scanned by an XY scanning mirror (not shown), so that the laser light
Only the fluorescent molecules on the focal plane irradiated with 3 are excited. The fluorescence from the excited fluorescent molecules is received by the photomultiplier tube 26 and image-processed by a computer, whereby a fluorescence image of the tomographic plane of the sample 21 is obtained. The image of each tomographic plane is obtained by changing the focal plane, and further image processing is performed.
A two-dimensional image can also be obtained.

The measurement using the free fluorescent molecules in the cell is slower than the above-described confocal laser microscope because the fluorescent molecules only at the focal plane are excited. The bleached fluorescent molecules on the focal plane are replaced by out-of-focus, unbleached fluorescent molecules, so that an image on the same plane is repeatedly recorded without bleaching.
Furthermore, even in the case of recording using fluorescent molecules fixed in a sample, a three-dimensional image can be recorded almost without fading since fluorescent molecules out of focus do not fade. In addition, since a laser having a wavelength about twice that of single-photon excitation (such as in the case of a confocal laser microscope) is used, laser scattering is small, laser light reaches deep inside tissue, and fluorescent light in a deep layer is emitted. An image is obtained. In addition, even when a fluorescent substance excited by ultraviolet light is used, photometry can be performed by an optical system in the visible light range, and cytotoxicity is low. The configuration of the two-photon excitation laser microscope in FIG. 6 is common to the configuration of a multiphoton excitation laser using excitation by a plurality of photons.

Now, when observing a sample using the above-mentioned microscope, the amount of light that can be generally used for observation is small because fluorescence is used. Moreover, in the microscope described above, of the fluorescent light generated in all directions from the sample, only the fluorescent light falling within the aperture angle of the objective lens 22 is used for observation.

[0014]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microscope for observing fluorescence by making use of the fluorescence generated by the sample so that the fluorescence can be used for observation. This is to increase the amount of light.

[0015]

SUMMARY OF THE INVENTION In order to achieve the above object, the present invention provides not only the fluorescence directed to the upper side (or lower side in an inverted microscope) of the sample but also the leakage to the lower (or upper) side of the sample. The system is designed to be able to use existing fluorescent light. Specifically, in a microscope for observing a sample with fluorescence generated from the sample, an objective lens for condensing the fluorescence generated from the sample, and the objective lens, the sample is disposed on the opposite side of the sample. And a reflecting mirror for reflecting the fluorescence generated from the sample, and observing the sample by the fluorescence by the reflection from the reflecting mirror and the direct fluorescence.

The reflecting mirror is a plane reflecting mirror and may be used together with a lens, or may be a concave reflecting mirror. The space between the concave reflecting mirror and the sample may be filled with water or oil. Further, the reflecting mirror may be placed on an optical path via an optical path changing device. The microscope to be applied includes an epifluorescence microscope, a confocal laser microscope, a multiphoton excitation laser microscope, and the like.

[0017]

Embodiments of the present invention will be described in detail with reference to the drawings. In each of the drawings, the same components are denoted by the same reference numerals. The present invention is characterized in that not only the fluorescence directed to the upper side (or lower side) of the sample but also the fluorescence leaking to the lower part (upper part) of the sample can be used for observation of the sample.

(Embodiment 1) In Embodiment 1 of the present invention, a plane mirror and a lens are used in order to utilize fluorescence in a lower direction of a sample. The configuration will be described in detail with reference to FIGS. FIG. 7 shows a configuration in an epi-fluorescence microscope. 7, the description of the same configuration as that of the epi-illumination fluorescence microscope shown in FIG. 1 is omitted.

Now, with the epi-fluorescence microscope shown in FIG.
An afocal objective lens 42 is placed in the space on the opposite side of the sample glass 21 via the cover glass 40, and the focal point of the objective lens 42 matches the focal point of the objective lens 22. Objective lens 42
Is emitted from the plane mirror 4 as parallel rays.
The light is reflected at 4 and coincides with the optical path of the objective lens 22. With such a configuration, it is possible to form an image with the objective lens 22 by combining the fluorescence directly emitted from the sample 21 and the fluorescence reflected by the plane mirror 44. as a result,
A bright fluorescent image in which the fluorescence intensity is enhanced by the eyepiece lens 25 can be observed.

The configuration using such a plane mirror and a lens can be applied to the confocal laser microscope and the multiphoton excitation laser microscope described with reference to FIGS. FIG. 8 and FIG. 9 illustrate this. FIG.
Shows the configuration of a confocal laser microscope, and FIG. 9 shows the configuration of a multiphoton excitation laser microscope. 8 and 9, the description of the same configuration as that of the laser microscope shown in FIGS. 4 and 6 will be omitted.

In FIGS. 8 and 9, an afocal objective lens 42 is placed in a space on the opposite side of the sample 21 with a cover glass 40 interposed therebetween.
Match to the focus of 2. Since the light emitted from the objective lens 42 is a parallel light beam, this light beam is reflected by the plane mirror 44,
It is made to coincide with the optical path of the objective lens 22. With such a configuration, it is possible to measure the fluorescence enhanced by the detector 26 through the objective lens 22 by combining the fluorescence directly emitted from the sample 21 and the fluorescence reflected by the plane mirror 44.

In the configuration shown in FIGS. 7 to 9, the plane mirror 44 is mounted on an angle changing device for adjusting the optical axis, and the optical path can be changed and can be moved in and out of the optical path. Thus, the optical path can be changed to match the optical path of the objective lens 22, and can be removed from the optical path when the plane mirror 44 is not used. The above configuration can be applied to both upright and inverted microscopes. In an inverted microscope, the upper and lower parts in the above description are reversed with respect to the sample.

(Embodiment 2) In Embodiment 2 of the present invention, a concave mirror is used in order to utilize the fluorescence in the lower direction of the sample. The configuration will be described in detail with reference to FIGS. FIG. 10 shows a configuration in an epi-illumination fluorescence microscope. 10, description of the same configuration as that of the epi-illumination fluorescence microscope shown in FIGS. 1 and 7 is omitted.

In the epi-illumination fluorescence microscope shown in FIG. 10, a concave reflecting mirror 46 is placed in the space on the opposite side of the sample glass 21 via the cover glass 40, and the focal point of the concave reflecting mirror 46 matches the focal point of the objective lens 22. . The fluorescent light from the cover glass 40 is reflected by the concave reflecting mirror 46 so as to match the optical path of the objective lens 22. With such a configuration, it is possible to form an image with the objective lens 22 by combining the fluorescence directly emitted from the sample 21 and the fluorescence reflected by the concave reflecting mirror 46. As a result, a bright fluorescent image in which the fluorescence intensity is enhanced by the eyepiece lens 25 can be observed.

The configuration using such a concave reflecting mirror can be applied to the confocal laser microscope and the multi-photon excitation laser microscope described with reference to FIGS. This is illustrated in FIGS. 11 and 12. FIG.
Shows the configuration of a confocal laser microscope, and FIG. 12 shows the configuration of a multiphoton excitation laser microscope. FIG. 11 and FIG.
In FIG. 2, the description of the same configuration as that of the laser scanning microscope shown in FIGS. 4 and 6 will be omitted.

11 and 12, a concave reflecting mirror 46 is placed in a space on the opposite side of the sample glass 21 via the cover glass 40, and the focal point of the concave reflecting mirror 46 matches the focal point of the objective lens 22. The fluorescent light from the cover glass 40 is reflected by the concave reflecting mirror 46 so as to match the optical path of the objective lens 22. With such a configuration, the fluorescence directly emitted from the sample 21 and the fluorescence reflected by the concave reflecting mirror 46 can be combined, and the fluorescence can be measured by the detector 26 via the objective lens 22.

In the configuration shown in FIGS. 10 to 12, the space between the concave reflecting mirror 46 and the cover glass 40 can be immersed in water or oil. In this case, the curved surface of the concave reflecting mirror 46 is a regular hemisphere, and does not depend on the characteristics of the objective lens 22. The expansion of the regular hemisphere is made to match the aperture angle of the objective lens 22. Concave reflector 46 and cover glass 4
0 is not immersed in water or oil, the concave mirror
6 changes the curved surface of the concave reflecting mirror 46 in accordance with the thickness of the objective lens 22 and the cover glass 40 and the like, and adjusts the expansion to the expansion of the objective lens 22.

Also, the concave reflecting mirror 46 can be mounted on an angle changing device for adjusting the optical axis, so that the optical path can be changed or can be moved in and out of the optical path. Thereby, it can be made to coincide with the optical path of the objective lens 22, and can be removed from the optical path when the concave reflecting mirror 46 is not used. The above configuration can be applied to both upright and inverted microscopes. In an inverted microscope, the upper and lower parts in the above description are reversed with respect to the sample.

[0029]

As described above, the present invention can be used for observing a sample not only of the fluorescent light directed to the upper side (lower side) of the sample but also the fluorescent light leaking to the lower part (upper part) of the sample. It has such a configuration. For this reason, a bright fluorescent image with enhanced fluorescent intensity can be observed.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a configuration of an epi-illumination fluorescence microscope.

FIG. 2 is a graph for explaining a dichroic mirror, an excitation filter, and an absorption filter that separate excitation light and fluorescence in an epi-illumination fluorescence microscope.

FIG. 3 is a diagram for explaining single-photon excitation.

FIG. 4 is a diagram illustrating a configuration of a confocal laser microscope.

FIG. 5 is a diagram for explaining two-photon excitation.

FIG. 6 is a diagram illustrating a configuration of a two-photon excitation laser microscope.

FIG. 7 is a diagram illustrating a configuration of an epi-illumination fluorescence microscope according to the first embodiment.

FIG. 8 is a diagram illustrating a configuration of a multiphoton excitation laser microscope according to the first embodiment.

FIG. 9 is a diagram illustrating a configuration of a multiphoton excitation laser microscope according to the first embodiment.

FIG. 10 is a diagram illustrating a configuration of an epi-illumination fluorescence microscope according to a second embodiment.

FIG. 11 is a diagram illustrating a configuration of a multiphoton excitation laser microscope according to the first embodiment.

FIG. 12 is a diagram illustrating a configuration of a multiphoton excitation laser microscope according to a second embodiment.

[Explanation of symbols]

 Reference Signs List 10 adiabatic potential curve 11 adiabatic potential curve 21 sample 22 objective lens 23 laser beam 24 dichroic mirror 25 lens 25 eyepiece 26 detector 27 confocal pinhole 32 irradiation light 34 excitation filter 36 absorption filter 40 cover glass 42 objective lens 44 plane mirror 46 Concave reflector

Claims (8)

[Claims] 1. A microscope for observing a sample with fluorescence generated from the sample, comprising: an objective lens for condensing fluorescence generated from the sample; and a sample on the opposite side of the sample from the objective lens. A reflecting mirror for reflecting the fluorescence generated from the mirror, and observing the sample with the fluorescence due to the reflection from the reflecting mirror and the direct fluorescence. 2. The microscope according to claim 1, wherein the reflecting mirror is a plane reflecting mirror and used together with a lens. 3. The microscope according to claim 1, wherein said reflecting mirror is a concave reflecting mirror. 4. The microscope according to claim 3, wherein a space between the concave reflecting mirror and the sample is filled with water or oil. 5. The microscope according to claim 1, wherein the reflecting mirror is mounted on an optical path changing device. 6. The microscope according to claim 1, wherein the microscope is an epi-illumination fluorescence microscope. 7. The microscope according to claim 1, wherein the microscope is a confocal laser microscope. 8. The microscope according to claim 1, wherein the microscope is a multiphoton excitation laser microscope.