JP2003270538A - Microscope system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope system which can easily deal with changeover of a plurality of kinds of different observation methods or simultaneous use thereof. <P>SOLUTION: The microscope system has a laser beam source (1), an optical path splitting section (2) for splitting the optical path of the laser beam from the laser beam source to at least two, a plurality of optical fibers (3 and 4) respectively made incident with the laser beams on the optical path split in the optical path splitting section and a plurality of different microscope optical systems respectively using the laser beams part a plurality of the optical fibers. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope system capable of coping with a plurality of different observation methods.

[0002]

2. Description of the Related Art Recently, functional analysis of living cells has been conducted. In the functional analysis of these cells, in particular, in order to observe the function of the cell membrane, a total reflection fluorescence microscope that obtains an evanescent fluorescence image from the cell membrane and its vicinity has attracted attention.

Japanese Unexamined Patent Publication No. 10-231222 discloses an example of a scanning laser microscope. In this scanning laser microscope, to support multiple fluorescent dyes,
The laser beams oscillated from multiple laser light sources are combined into one laser beam, and the sample is irradiated while scanning the sample through the objective lens as a point light source, and the observation light of fluorescence or reflected light from the sample is again objective lens. A two-dimensional information is obtained by detecting with a photodetector. By utilizing the confocal effect, such a scanning laser microscope obtains only the information on the focused surface, and is used as an effective observation method for slice observation of a thick sample.

On the other hand, Japanese Patent Laid-Open No. 2001-272606 shows an example of a total reflection fluorescence microscope. In this total reflection fluorescence microscope, the laser beam oscillated from the laser light source is focused from the exit end face of the optical fiber to the rear focal point of the objective lens through the focusing optical system, and the laser beam from the objective lens is radiated. By illuminating the specimen by injecting it with the axis inclined, fluorescence observation with evanescent light is possible. In such a total reflection fluorescence microscope, the illumination range is limited to the depth of about the wavelength of the laser used as the light source, so the background fluorescence is very small, and the observation of the cell membrane surface and the localization near the cover glass surface are localized. It is used as an effective observation method for observing one molecule of a fluorescent dye.

[0005]

However, these scanning laser microscopes and total reflection fluorescence microscopes use optical fibers, but each microscope is compatible with only one observation method. Therefore, for example, when observing fluorescence observation of one specimen labeled with a fluorescent dye using a confocal scanning laser microscope and a total reflection fluorescence microscope, the specimen must be moved between the microscopes. I won't. Therefore, particularly when observing the state of the same position on the sample, there is a problem in that it is extremely difficult for each device to find the same position on the sample.

It is an object of the present invention to provide a microscope system which can easily handle switching or simultaneous use of a plurality of different observation methods.

[0007]

In order to solve the problems and achieve the object, the microscope system of the present invention is configured as follows.

In the microscope system of the present invention, a laser light source, an optical path splitting section for splitting the optical path of the laser beam from the laser light source into at least two, and each laser beam on the optical path split by the optical path splitting section are A single microscope includes a plurality of incident optical fibers and a plurality of different microscope optical systems that respectively use the laser beams that have passed through the plurality of optical fibers.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 is a diagram showing a schematic configuration of a microscope system according to a first embodiment of the present invention. On the optical path of the laser beam emitted from the laser light source 1, a beam splitter 2 for beam splitting is arranged as an optical path splitting member. The beam splitter 2 splits the laser beam emitted from the laser light source 1 so that 50% is transmitted and 50% is reflected.

On the reflected light path of the beam splitter 2, a shutter 25 as a light shielding member, a condenser lens 301,
The entrance end 3a of the single mode optical fiber 3 is arranged. The laser beam reflected by the beam splitter 2 and condensed by the condenser lens 301 has an incident end 3a.
Is incident on. The shutter 25 is opened and closed under the control of the control unit 100.

On the transmission optical path of the beam splitter 2, a shutter 26 as a light shielding member and a condenser lens 3 are provided.
02, and the entrance end 4 of the single mode optical fiber 4
a is arranged. The laser beam transmitted through the beam splitter 2 and condensed by the condenser lens 302 is incident on the incident end 4a. The shutter 26 is opened and closed under the control of the control unit 100.

The shutters 25 and 26 are laser light sources 1.
It blocks the laser beam emitted from. When the microscope system of FIG. 1 is used as a confocal scanning laser microscope, the shutter 25 is opened and the shutter 26 is opened.
Is closed. When used as a total reflection fluorescence microscope, the shutter 25 is closed and the shutter 26 is opened. Both shutters 25, 26 are closed when not used as either microscope.

On the optical path of the output end 3b of the optical fiber 3,
Confocal scanning laser microscope (L
An optical system (microscope optical system) that constitutes SM) is arranged. In this optical system, a collimator lens 5 and a dichroic mirror 6 are arranged on the optical path of the laser beam emitted from the emission end 3b of the optical fiber 3. An XY scanner 7, a pupil projection lens 8, and a mirror 9 are arranged on the reflection optical path of the dichroic mirror 6. Further, on the reflection optical path of the mirror 9, the imaging lens 1
0, an objective lens 11 forming an observation optical system, and a sample 12 are arranged. The mirror 9 can be inserted into and removed from the optical path by an insertion / removal mechanism 201 having an actuator. The insertion / removal mechanism 201 is driven under the control of the control unit 100.

The laser beam emitted from the emission end 3b of the optical fiber 3 is converted into a parallel light beam by the collimator lens 5, reflected by the dichroic mirror 6, and the laser beam emitted from the XY scanner 7 passes through the pupil projection lens 8. It passes through and is reflected by the mirror 9, passes through the imaging lens 10, is focused on the sample 12 by the objective lens 11, and is scanned. In this case, the mirror 9 is inserted in the optical path.

The fluorescence and reflected light from the sample 12 follow the above-mentioned optical paths in reverse, that is, the objective lens 11, the imaging lens 10, the mirror 9, the pupil projection lens 8, and the XY.
It is incident on the dichroic mirror 6 via the scanner 7.

On the transmission optical path of the dichroic mirror 6, the mirrors 401 and 402 and the dichroic mirror 13 are provided.
a and the mirror 13b are arranged. The dichroic mirror 6 transmits the fluorescence having a longer wavelength than the laser beam and reflects the laser beam which is the reflected light from the sample 12. Specimen 1 that passed through dichroic mirror 6
Fluorescence from 2 is split by the dichroic mirror 13a for each wavelength via the mirrors 401 and 402.

An absorption filter 14a and a confocal lens 15 are provided on the reflection optical path of the dichroic mirror 13a.
a, a confocal diaphragm 16a provided at a position optically conjugate with the sample 12, and a detector 17a are arranged. The fluorescence from the sample 12 that has passed through the absorption filter 14a is condensed by the confocal lens 15a, and the confocal diaphragm 16
After passing through a, it is detected by the detector 17a.

Similarly, on the reflection optical path of the mirror 13b,
The confocal diaphragm 1 provided at a position optically conjugate with the absorption filter 14b, the confocal lens 15b, and the sample 12.
6b and a detector 17b are arranged. The fluorescence from the sample 12 that has passed through the dichroic mirror 13a is
The confocal lens 1 passes through the absorption filter 14b.
The light is condensed by 5b, passes through the confocal diaphragm 16b, and then the detector 1
It is detected at 7b.

On the other hand, an optical system (microscope optical system) forming a total reflection fluorescence microscope as a second observation method is arranged on the optical path of the emission end 4b of the optical fiber 4. In this optical system, the output end 4b of the optical fiber 4 is connected to the objective lens 11
It is arranged so as to be conjugate with the rear focal position 18. Further, the optical system 21 in the epi-illumination light projection tube 21 is provided on the optical path of the laser beam emitted from the emission end 4b of the optical fiber 4.
a and the dichroic mirror 22 are arranged. The laser beam emitted from the emission end 4b passes through the optical system 21a in the epi-illumination projection tube 21, is reflected by the dichroic mirror 22 toward the objective lens 11, and is collected at the rear focal position 18 of the objective lens 11. Be illuminated. The dichroic mirror 22 can be inserted into and removed from the optical path by an insertion / removal mechanism 202 including an actuator. The insertion / removal mechanism 202 is driven under the control of the control unit 100.

The emitting end 4b of the optical fiber 4 can be moved in a direction perpendicular to the optical axis 20 of the optical system 21a by a moving mechanism 203 having an actuator. Moving mechanism 20
3 is driven by the control of the control unit 100. The position of the output end 4b of the optical fiber 4 is moved in the direction perpendicular to the optical axis 20 so that the parallel light flux emitted from the objective lens 11 is inclined and incident on the sample 12, and the incident angle at this time is changed. Adjust to the specified angle. As a result, the sample 12
Alternatively, total reflection occurs at the boundary of the cover glass, and a very small part of the laser beam seeps out to the sample 12 side as evanescent light (evanescent field).

The fluorescence near the boundary of the sample 12 due to the evanescent light passes through the objective lens 11, the dichroic mirror 22, the imaging lens 10, and the absorption filter 23, and is imaged by the image pickup device 24 (for example, CCD camera). . In this case, the mirror 9 is removed from the optical path.

The dichroic mirror 22 of the first embodiment is used when the sample 12 is dyed with a monochromatic fluorescent dye, or when the sample 12 is multi-stained and only the monochromatic fluorescent dye is observed. , The second observation method, that is, the characteristics necessary for performing fluorescence observation with evanescent light. That is, the dichroic mirror 22
The laser beam having the excitation wavelength emitted from the emission end 4b of the optical fiber 4 is reflected, and fluorescence from the sample 12 (emission end 4
It has a wavelength characteristic of transmitting fluorescence (excited by a laser beam of the excitation wavelength emitted from b).

When the microscope system configured as described above is used as a confocal scanning laser microscope, the shutter 25 is opened and the shutter 26 is closed.
The mirror 9 is inserted into the optical path by the insertion / removal mechanism 201.
The dichroic mirror 22 is removed from the optical path by the insertion / removal mechanism 202. In this state, the laser beam emitted from the laser light source 1 is reflected by the beam splitter 2 and passes through the condenser lens 301 to enter the incident end 3 of the optical fiber 3.
It is incident on a. The laser beam emitted from the emission end 3b of the optical fiber 3 is converted into a parallel light flux by the collimator lens 5, reflected by the dichroic mirror 6, further passed through the XY scanner 7 and the pupil projection lens 8, and then by the mirror 9. The light is reflected, passes through the imaging lens 10, is focused on the sample 12 by the objective lens 11, and is scanned.

The fluorescence and the reflected light from the sample 12 follow the above-mentioned optical paths in reverse, and only the fluorescence having a longer wavelength than the laser beam passes through the dichroic mirror 6, and passes through the mirrors 401 and 402 for each wavelength. The light is split by the dichroic mirror 13a. Dichroic mirror 13
The fluorescence in the wavelength region reflected by a is absorbed by the absorption filter 14
a, the confocal lens 15a, and the confocal diaphragm 1
It is detected by the detector 17a via 6a and is acquired as a two-dimensional image. At the same time, the fluorescence in the wavelength region transmitted through the dichroic mirror 13a is detected by the detector 17b via the mirror 13b, the absorption filter 14b, the confocal lens 15b, and the confocal diaphragm 16b, and is acquired as a two-dimensional image.

On the other hand, when the microscope system is used as a total reflection fluorescence microscope, the shutter 25 is closed and the shutter 26 is opened. The mirror 9 is removed from the optical path by the insertion / removal mechanism 201. The dichroic mirror 22 is inserted into the optical path by the insertion / removal mechanism 202. In this state, the laser beam emitted from the laser light source 1 is
The light passes through the beam splitter 2 and enters the incident end 4 a of the optical fiber 4 via the condenser lens 302. Further, the laser beam emitted from the emission end 4 b of the optical fiber 4 passes through the optical system 21 a in the epi-illumination light projection tube 21, is reflected by the dichroic mirror 22 toward the objective lens 11, and after the objective lens 11. The light is focused on the side focal position 18.

In this state, the output end 4b of the optical fiber 4 is
Is moved in the direction perpendicular to the optical axis 20 of the optical system 21a by the moving mechanism 203, and the incident angle of the laser light obliquely incident on the sample 12 from the objective lens 11 is adjusted.
As a result, total reflection is generated at the boundary of the sample 12 or the cover glass, and the evanescent light is generated so as to exude to the sample 12 side. The fluorescence near the boundary of the sample 12 due to the evanescent light passes through the objective lens 11, the dichroic mirror 22, the imaging lens 10, and the absorption filter 23, and is imaged by the image sensor 24 (for example, CCD camera).

When the microscope system is not used as a confocal scanning laser microscope or a total reflection fluorescence microscope, both shutters 25 and 26 are closed. By doing so, it is possible to prevent the specimen 12 from being discolored by the laser beam.

According to the first embodiment, the observation optical system constituted by the objective lens 11 is commonly used regardless of whether the microscope system is used as a confocal scanning laser microscope or a total reflection fluorescence microscope. To be Therefore, for example, even when the fluorescence from one specimen 12 labeled with a fluorescent dye is observed by using the observation method of both the confocal scanning laser microscope and the total reflection fluorescence microscope, the specimen 12 is separated. No need to move to microscope.
Thus, when the state of the same position on the sample 12 is observed by different observation methods, highly accurate observation can be performed simply by switching the observation method with the same microscope system.

Conventionally, the confocal scanning laser microscope and the total reflection fluorescence microscope have been used as separate microscopes. In this case, a laser light source having a laser beam of the same wavelength is required for each microscope, but one set of this laser light source must be prepared for each microscope, which is extremely economically disadvantageous. Therefore,
It is also possible to share one laser light source with each microscope. In this case, the optical fiber from the laser source,
Although it will be reconnected for each microscope to be used, readjustment of the optical axis is necessary in order to guide the laser light to the sample as illumination. However, the optical fibers used in these scanning laser microscopes and total reflection fluorescence microscopes are usually single mode fibers, and the core diameter is extremely small, such as several microns, so it is extremely difficult for the user to adjust the optical axis.

On the other hand, in the first embodiment, the laser beam from the laser light source 1 split by the beam splitter 2 is passed through the optical fibers 3 and 4, respectively, and the confocal scanning laser microscope and total reflection are used. It is introduced into an optical system that constitutes a fluorescence microscope and used as a light source for each observation method. As a result, the laser light source can be shared as compared with the case where one set of laser light source is prepared for each microscope as described above, so that the entire microscope system can be downsized and it is extremely economically advantageous.

Further, in the first embodiment, the position of the beam splitter 2 with respect to the laser light source 1 is fixed, and the laser beam with respect to the incident ends 3a and 4a of the optical fibers 3 and 4 when the observation method is switched. There is no displacement. Therefore, as described above, these difficult operations can be omitted and a stable amount of light can be constantly supplied to each microscope, as compared with the one that requires readjustment of the optical axis every time the observation method is switched. .

Furthermore, since a confocal scanning laser microscope and a total reflection fluorescence microscope, that is, optical systems for different observation purposes can be provided as one system,
You can easily adapt to the observation method that suits your purpose. Further, since the laser light source and the observation optical system can be commonly used for each observation method, the system space can be reduced, the degree of freedom in layout can be increased, and the cost can be reduced.

(Second Embodiment) FIG. 2 is a diagram showing a schematic configuration of a microscope system according to a second embodiment of the present invention. 2, the same parts as those in FIG. 1 are designated by the same reference numerals.

A mirror 27 is arranged on the optical path of the laser beam emitted from the laser light source 1. This mirror 27 includes an insertion / removal mechanism 204 including an actuator.
Thus, the laser light source 1 can be moved in the direction perpendicular to the optical axis of the laser light source 1 (the direction of the arrow in the drawing), that is, can be inserted into and removed from the optical path. The insertion / removal mechanism 204 is driven by the control of the control unit 100. In the state where the mirror 27 is inserted in the optical path, the laser beam from the laser light source 1 is reflected by the mirror 27 and passes through the condenser lens 301 to the incident end 3 of the optical fiber 3.
It is incident on a. Further, when the mirror 27 is removed from the optical path, the laser beam from the laser light source 1 is directly incident on the incident end 4 a of the optical fiber 4 via the condenser lens 302. Other configurations in FIG. 2 are the same as those in FIG.

The dichroic mirror 22 of the second embodiment has the same characteristics as those of the first embodiment.

According to the second embodiment, the mirror 27
Is inserted into the optical path, the laser beam from the laser light source 1 is introduced into the incident end 3a of the optical fiber 3, so that the laser light source 1 is used as the light source of the confocal scanning laser microscope described in the first embodiment. be able to. Further, when the mirror 27 is removed from the optical path, the laser beam from the laser light source 1 is directly introduced to the incident end 4a of the optical fiber 4, so that the laser light source 1 can be used in the total reflection fluorescence microscope described in the first embodiment. It can be used as a light source.

Therefore, by using such a movable mirror 27, one laser light source can be switched and used as a light source for two different observation methods. Further, since the optical path dividing member for dividing the laser beam from the laser light source is not used, the laser beam can be used for each observation method without reducing its intensity.

(Third Embodiment) FIG. 3 is a diagram showing a schematic configuration of a microscope system according to a third embodiment of the present invention. In FIG. 3, the same parts as those in FIGS. 1 and 2 are designated by the same reference numerals.

In FIG. 3, a laser light source 29 having a multi-wavelength oscillation line and a laser light source 30 having a single-wavelength oscillation line are used as the laser light sources. The laser light source 29 is, for example, an argon laser, and is 457.9.
nm, 488 nm, and 514.5 nm. The laser light source 30 is, for example, a helium neon laser and has an oscillation line of 543 nm.

On the optical path of the laser beam emitted from the laser light source 29, a dichroic mirror 31 for beam combining as an optical path combining member and a beam splitter 2 for beam splitting are arranged. A mirror 30 is provided on the optical path of the laser beam emitted from the laser light source 30.
4 are arranged. The dichroic mirror 31 synthesizes the laser beams from the laser light sources 29 and 30,
It is incident on the beam splitter 2 via one optical path.

On the reflection optical path of the beam splitter 2, the AOTF is provided between the beam splitter 2 and the condenser lens 301.
An (acousto-optic variable wavelength filter) 32 is arranged.
Further, an AOTF 33 is arranged on the transmission optical path of the beam splitter 2 between the beam splitter 2 and the condenser lens 302. The AOTFs 32 and 33 act as selection filters for a plurality of oscillation lines. AOTF32,
The operation of 33 is controlled by the control unit 100. AO
By selectively applying the RF voltage to each of the TFs 32 and 33, the oscillation line corresponding to the frequency is selected as the oscillation line to be introduced into each of the optical fibers 3 and 4. As a result, simultaneous observation by laser beams of different wavelengths can be realized by the confocal scanning laser microscope and the total reflection fluorescence microscope.

The dichroic mirror 22 of the third embodiment uses the first observation method, that is, the fluorescence observation by the LSM and the second observation, by using the laser beams of different excitation wavelengths when the sample 12 is multi-stained. It has the characteristics necessary for simultaneously performing the observation method (i.e., fluorescence observation with evanescent light). That is, the dichroic mirror 22 reflects the laser beam of the excitation wavelength emitted from the emission end 4b of the optical fiber 4, and the fluorescence from the sample 12 (the fluorescence excited by the laser beam of the excitation wavelength emitted from the emission end 4b). Has a wavelength characteristic of transmitting the laser beam of the excitation wavelength emitted from the emission end 3b of the optical fiber 3, and transmits the fluorescence from the sample 12 (excited by the laser beam of the excitation wavelength emitted from the emission end 3b). Has a wavelength characteristic of transmitting fluorescence.
Other configurations in FIG. 3 are the same as those in FIG.

When the microscope system configured as described above is used as a confocal scanning laser microscope, the mirror 9 is inserted into the optical path by the insertion / removal mechanism 201. The dichroic mirror 22 is removed from the optical path by the insertion / removal mechanism 202. In this state, laser beams emitted from the laser light sources 29 and 30 (457.9 nm, 488 nm,
514.5 nm and 543 nm oscillation lines) are AOT
An oscillation line having a wavelength of 488 nm is selected by F32, and the sample 12 is irradiated through the optical fiber 3 or the like. At this time,
In the AOTF 33, the oscillation line having the wavelength of 488 nm is not selected. Then, FITC (fluorescence wavelength of about 520 nm) and T
In the sample 12 that has been multi-stained with RITC (fluorescence wavelength of about 585 nm), fluorescence corresponding to an oscillation line (excitation light) having a wavelength of 488 nm is excited.

The beam containing the fluorescence of FITC having a wavelength of less than 560 nm from the sample 12 is reflected by the mirror 9 toward the optical path side for the confocal scanning laser microscope. FITC
Of fluorescence is detected by the detectors 17a and 17b.

When the microscope system configured as described above is used as a total reflection fluorescence microscope, the mirror 9 is removed from the optical path by the insertion / removal mechanism 201. The dichroic mirror 22 is inserted into the optical path by the insertion / removal mechanism 202. In this state, laser beams (457.9 nm, 488 nm, 514.5 nm) emitted from the laser light sources 29, 30 are emitted.
nm and 543 nm oscillation line), the oscillation line having a wavelength of 543 nm is selected by the AOTF 33, and the sample 12 is irradiated through the optical fiber 4 or the like. At this time, AOTF3
In 2, the oscillation line with a wavelength of 543 nm is not selected. FITC (fluorescence wavelength of about 520 nm) and TRITC
Specimen 12 multi-stained with (fluorescence wavelength of about 585 nm)
In, the fluorescence corresponding to the oscillation line (excitation light) having a wavelength of 643 nm is excited.

The beam containing the fluorescence of TRITC having a wavelength of 560 nm or more from the sample 12 is detected by the image pickup device 24.

When the microscope system having the above-mentioned configuration is used simultaneously as a confocal scanning laser microscope and a total reflection fluorescence microscope, a dichroic mirror 9'is used instead of the mirror 9 described above. The dichroic mirror 9'reflects a beam having a wavelength of less than 560 nm and transmits a beam having a wavelength of more than that. The dichroic mirror 9 ′ is inserted into the optical path by the insertion / removal mechanism 201. The dichroic mirror 22 is inserted into the optical path by the insertion / removal mechanism 202.

In this state, laser beams (457.9 nm, 488n) emitted from the laser light sources 29, 30 are emitted.
m, 514.5 nm, and 543 nm oscillation line) is A
An oscillation line having a wavelength of 488 nm is selected for the confocal scanning laser microscope by the OTF 32, and this oscillation line is reflected by the dichroic mirror 9 ′ via the optical fiber 3 or the like, transmitted through the dichroic mirror 22, and irradiated onto the sample 12. At the same time, an oscillation line having a wavelength of 543 nm is selected by the AOTF 33 for the total reflection fluorescence microscope, and this oscillation line is transmitted through the optical fiber 4 or the like to the dichroic mirror 2.
It is reflected by 2 and is irradiated to the sample 12. And FITC
(Fluorescence wavelength of about 520 nm) and TRITC (fluorescence wavelength of 5
Fluorescence corresponding to each oscillation line (excitation light) is excited in the sample 12 that is multi-stained with (about 85 nm).

These fluorescences from the sample 12 pass through the dichroic mirror 22 and the dichroic mirror 9 '.
Is separated by. The beam containing fluorescence of FITC having a wavelength of less than 560 nm is reflected by the dichroic mirror 9'to the optical path side for the confocal scanning laser microscope. 56
A beam containing TRITC fluorescence having a wavelength of 0 nm or more passes through the dichroic mirror 9'to the optical path side for a total reflection fluorescence microscope. The fluorescence thus separated is detected by the detector 1
7a, 17b and the image pickup device 24 simultaneously detect.

According to the third embodiment, a plurality of laser light sources are provided so that the oscillation line can be selectively used, and the confocal scanning laser microscope and the total reflection fluorescence microscope emit laser beams of different wavelengths. Can be used individually or simultaneously. This allows simultaneous observation of multiple-stained specimens.

(Fourth Embodiment) FIG. 4 is a diagram showing a schematic configuration of a microscope system according to a fourth embodiment of the present invention. 4, the same parts as those in FIGS. 1 to 3 are designated by the same reference numerals.

In FIG. 4, as in FIG. 3, a laser light source (argon laser) 29 having a multi-wavelength oscillation line and a single wavelength laser light source (helium neon laser) 30 are used as laser light sources.

A dichroic mirror 34 for beam combining is arranged on the optical path of the laser beam emitted from the laser light source 29. A mirror 304 is arranged on the optical path of the laser beam emitted from the laser light source 30. The dichroic mirror 34 beam-combines the laser beams from the laser light sources 29 and 30,
It is incident on the beam splitter 35.

A beam splitter 35 is arranged on the optical path of the beam combined by the dichroic mirror 34. The beam splitter 35 transmits 30% of the laser beam from the dichroic mirror 34,
Divide to reflect 70%. Beam splitter 35
AOTF 32, a condenser lens 301, and
The entrance end 3a of the single mode optical fiber 3 is arranged. The laser beam transmitted through the beam splitter 35 enters the AOTF 32. The laser beam of the wavelength selected by the AOTF 32 is incident on the incident end 3a of the optical fiber 3 via the condenser lens 301.

On the reflected light path of the beam splitter 35,
A beam splitter 36 and a mirror 361 are arranged. This beam splitter 36 is a beam splitter 3
The laser beam from 5 is split so that 50% is transmitted and 50% is reflected. The AOTF 33, the condenser lens 302, and the incident end 4 a of the single-mode optical fiber 4 are arranged on the reflection optical path of the beam splitter 36. The laser beam reflected by the beam splitter 36 enters the AOTF 33. The laser beam of the wavelength selected by the AOTF 33 is incident on the incident end 4a of the optical fiber 4 via the condenser lens 302.

AOTF is provided on the reflection optical path of the mirror 361.
37, the condenser lens 303, and the incident end 38a of the optical fiber 38 are arranged. The laser beam reflected by the beam splitter 361 enters the AOTF 37. A
The laser beam of the wavelength selected by the OTF 37 is incident on the incident end 38a of the optical fiber 38 via the condenser lens 303. The operation of AOTF 32, 33, 37 is
It is controlled by the control unit 100.

On the optical path of the emission end 38b of the optical fiber 38, an optical system (microscope optical system) which constitutes a confocal disc scanning microscope by a confocal scanner is arranged as a third observation method. This confocal scanner is an optical scanner that utilizes the confocal effect of multi-pinholes, and enables observation of slice images of a sample in real time.

On the optical path of the laser beam emitted from the emission end 38b of the optical fiber 38, a microlens array disk having a beam expander 39 for enlarging the diameter of the laser beam to a predetermined diameter and forming a parallel light flux, and a microlens 40a. 40 and a pinhole disc 41 having a pinhole 41a at a position optically conjugate with the specimen 12.
Are arranged. For the microlens array disk 40 and the pinhole disk 41, for example, a microlens array disk and Nipkow disk as disclosed in JP-A-9-80315 can be used. Further, instead of the pinhole disc 41, a striped disc (slit disc) as shown in International Publication No. WO01 / 67155 can be used.

The laser beam emitted from the emission end 38b of the optical fiber 38 has its beam diameter expanded by the beam expander 39 to a predetermined diameter, and is converted into a parallel light beam. The laser beam converted into the parallel light flux passes through the microlens 40a of the microlens array disk 40,
The light is focused on the pinhole 41a on the pinhole disk 41 arranged corresponding to the microlens 40a. The microlens array disk 40 and the pinhole disk 41 rotate integrally around a common shaft 42.

Pinhole 41 of pinhole disk 41
An imaging lens 10, an objective lens 11, and a sample 12 are arranged on the optical path of the laser beam passing through a. When the confocal scanner is used, the mirror 9 and the mirror 46 are removed from the optical path as described later. The laser beam that has passed through the pinhole 41 a of the pinhole disc 41 is formed by the imaging lens 10 and the objective lens 1.
After passing through 1, the light is focused on the sample 12.

The fluorescent light and the reflected light from the sample 12 pass through the objective lens 11 and the imaging lens 10 and reach the pinhole 41a on the pinhole disk 41. At this time,
Since the pinhole 41a is arranged at a position optically conjugate with the sample 12, the pinhole 41a provides a confocal effect.

A dichroic mirror 43 is arranged between the microlens array disc 40 and the pinhole disc 41. The light beam that has passed through the pinhole 41a is separated by the dichroic mirror 43. The dichroic mirror 43 reflects only the fluorescence from the sample 12. Further, on the reflection optical path of the dichroic mirror 43, the condenser lens 44 and the image pickup device (for example, CCD
A camera) 45 is arranged. The fluorescence from the sample 12 reflected by the dichroic mirror 43 is collected by the condenser lens 44.
A confocal image is picked up on the image pickup surface of the image pickup element 45 via the.

Further, a mirror 46 is arranged on the optical path between the imaging lens 10 and the mirror 9. Mirror 4
6 can be inserted into and removed from the optical path by an insertion / removal mechanism 205 equipped with an actuator. The insertion / removal mechanism 205 is driven by the control of the control unit 100. When the microscope system is used as a total reflection fluorescence microscope, this mirror 46 is inserted into the optical path by the insertion / removal mechanism 205 and reflects fluorescence near the boundary of the sample 12 due to the evanescent light.
The fluorescence reflected by the mirror 46 is imaged by the image sensor 24 (for example, CCD camera) via the absorption filter 23.

The mirror 46 is removed from the optical path by the insertion / removal mechanism 205 when the microscope system is used as a confocal scanning laser microscope or when a confocal scanner is used. The mirror 9 is an insertion / removal mechanism 2 when the microscope system is used as a confocal scanning laser microscope.
01 is inserted in the optical path, and when the confocal scanner is used, it is removed from the optical path by the insertion / removal mechanism 201.

As described above, the mirror 9 and the mirror 46 are appropriately inserted into and removed from the optical path according to the observation method used in the microscope system. This allows the microscope system to be used as a confocal scanning laser microscope, a total internal reflection fluorescence microscope, or a confocal scanner.

The dichroic mirror 22 of the fourth embodiment uses the first observation method, that is, the fluorescence observation by LSM and the second observation by using the laser beams of different excitation wavelengths when the sample 12 is multiply stained. It has the characteristics necessary for simultaneously performing the observation method (i.e., fluorescence observation with evanescent light) and the third observation method (e.g., fluorescence observation with a confocal scanner). That is, the dichroic mirror 22 reflects the laser beam of the excitation wavelength emitted from the emission end 4b of the optical fiber 4, and the fluorescence from the sample 12 (the fluorescence excited by the laser beam of the excitation wavelength emitted from the emission end 4b). Has a wavelength characteristic of transmitting the laser beam of the excitation wavelength emitted from the emission end 3b of the optical fiber 3, and transmits the fluorescence from the sample 12 (excited by the laser beam of the excitation wavelength emitted from the emission end 3b). Laser having an excitation wavelength emitted from the emission end 38b of the optical fiber 38 and transmitting fluorescence from the sample 12 (excitation wavelength emitted from the emission end 38b). It has a wavelength characteristic of transmitting fluorescence excited by a beam). Therefore, with the dichroic mirror 22 always inserted in the optical path, the microscope system can be used as a confocal scanning laser microscope, a total reflection fluorescence microscope, or a confocal scanner. Other configurations in FIG. 4 are the same as those in FIG.
Is the same as.

The mirrors 9 and 46 may be replaced with predetermined dichroic mirrors (not shown). The dichroic mirror that replaces the mirror 9 reflects the laser beam and fluorescence associated with the confocal scanning laser microscope and transmits the laser beam and fluorescence associated with the confocal scanner. The dichroic mirror, which replaces the mirror 46, reflects the fluorescence associated with the total internal reflection fluorescence microscope and transmits the laser beam and the fluorescence associated with the confocal scanning laser microscope and the confocal scanner. This enables simultaneous observation of each observation image.

According to the fourth embodiment, the laser beams from the laser light sources 29 and 30 are split into three laser beams and used as the light source for each observation method. In addition to the confocal scanning laser microscope and total internal reflection fluorescence microscope,
Furthermore, since a confocal scanner can be provided as an optical system for different observation purposes, it is possible to easily adapt to an observation method suitable for the purpose. As a result, since the laser light source can be shared, the system space can be reduced, the degree of freedom in layout can be increased, and the cost can be reduced. Further, similarly to the third embodiment, since laser beams having different wavelengths can be simultaneously used as a light source for each observation method, it is possible to perform fluorescence observation for each different staining of the multiple-stained specimen.

According to the present invention, it is possible to use a common observation optical system for different observation methods. As a result, for example, when observing the fluorescence of one specimen labeled with a fluorescent dye using different observation methods, it is possible to perform highly accurate observation simply by switching the observation method with the same microscope system. You can

Further, according to the present invention, since a common laser light source can be used as a light source for different observation methods, the entire system can be downsized and it is economically advantageous.

Furthermore, according to the present invention, since the optical system of a plurality of different observation methods can be provided as one system, it is possible to easily cope with the observation method suitable for the purpose.

Furthermore, according to the present invention, since the light shielding member is provided in the optical path for introducing the laser beam from the beam splitter to the optical fiber, the laser beam can be introduced only to the optical system of the observation method actually used. It is possible to prevent discoloration of the sample due to unnecessary laser beams.

As described above, according to the present invention, it is possible to provide a microscope system which can easily cope with switching or simultaneous use of a plurality of different observation methods.

The present invention is not limited to the above-described embodiment, but can be variously modified at the stage of carrying out the invention without departing from the spirit of the invention.

In FIG. 1 of the first embodiment, the optical system constituting the confocal scanning laser microscope as the first observation method is arranged on the optical path of the emitting end 3b of the optical fiber 3, An optical system constituting a total reflection fluorescence microscope as a second observation method is arranged on the optical path of the emission end 4b of the fiber 4. For example, an optical system constituting a confocal scanning laser microscope as the first observation method is arranged on the optical path of the emitting end 3b of the optical fiber 3, and the optical system of the third end is provided on the optical path of the emitting end 4b of the optical fiber 4. An optical system that constitutes a confocal disc scanning microscope as the observation method may be arranged. Further, an optical system constituting a confocal disc scanning microscope as a third observation method is arranged on the optical path of the emission end 3b of the optical fiber 3, and the second optical path is formed on the optical path of the emission end 4b of the optical fiber 4. An optical system forming a total reflection fluorescence microscope as an observation method may be arranged.

The present invention is not limited to the above-mentioned respective embodiments, and can be carried out by appropriately modifying it within the scope of the invention.

[0078]

According to the present invention, it is possible to provide a microscope system capable of easily adapting to the switching or simultaneous use of a plurality of different observation methods.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of a microscope system according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of a microscope system according to a second embodiment of the present invention.

FIG. 3 is a diagram showing a schematic configuration of a microscope system according to a third embodiment of the present invention.

FIG. 4 is a diagram showing a schematic configuration of a microscope system according to a fourth embodiment of the present invention.

[Explanation of symbols]

1 ... Laser light source 2 ... Beam splitter 3, 4 ... Optical fiber 5 ... Collimating lens 6 ... Dichroic mirror 7
XY scanner 8 Pupil projection lens 9 Mirror 10 Imaging lens 1
DESCRIPTION OF SYMBOLS 1 ... Objective lens 12 ... Specimen 21 ... Epi-illumination light projection tube 22 ... Dichroic mirror 23 ... Absorption filter 24 ... Imaging device 25 ... Shutter 26 ... Shutter 27 ... Mirror 30 ... Laser light source 31 ... Dichroic mirror 32, 33, 37 ... AO
TF 34 ... Dichroic mirror 35, 36 ... Beam splitter 38 ... Optical fiber 39 ... Beam expander 40 ... Microlens array disk 41 ... Pinhole disk 42 ... Shaft 43 ... Dichroic mirror 44 ... Condenser lens 45
Image pickup element 46 Mirror 100 Control section 201, 202, 20
4, 205 ... Inserting / removing mechanism 203 ... Moving mechanism 301, 302, 303 ... Condensing lens 304 ... Mirror 401, 402 ... Mirror

Claims (10)

[Claims] 1. A laser light source, an optical path splitting unit that splits an optical path of a laser beam from the laser light source into at least two, and a plurality of laser beams on the optical paths split by the optical path splitting unit are respectively incident. And a plurality of different microscope optical systems that respectively use the laser beams that have passed through the plurality of optical fibers, and a plurality of different microscope optical systems in a single microscope. 2. The microscope system according to claim 1, wherein the plurality of different microscope optical systems are a confocal scanning laser microscope optical system and a total reflection fluorescence microscope optical system. 3. The microscope system according to claim 1, wherein the plurality of different microscope optical systems are a confocal scanning laser microscope optical system and a confocal disc scan microscope optical system. 4. The microscope system according to claim 1, wherein the plurality of different microscope optical systems are a confocal disc scan microscope optical system and a total reflection fluorescence microscope optical system. 5. The microscope system according to claim 1, wherein the optical path splitting unit is a beam splitter. 6. The microscope system according to claim 5, wherein the microscope includes a plurality of light shielding members for shielding each laser beam on the optical path divided by the optical path dividing unit. 7. The optical path splitting part includes: at least one reflecting member for reflecting a laser beam; and an inserting / removing part for inserting / removing the reflecting member into / from an optical path of a laser beam from the laser light source. The microscope system according to any one of claims 1 to 4, which is characterized in that 8. The microscope includes a plurality of selection members which are respectively provided between the laser light source and the plurality of optical fibers and which select a wavelength of a laser beam incident on each of the plurality of optical fibers. The microscope system according to any one of claims 1 to 4, wherein: 9. The laser light source emits a plurality of laser beams having different wavelengths, and further comprises an optical path synthesizing member for synthesizing a plurality of laser beams having different wavelengths from the laser light source, in the microscope. The microscope system according to any one of claims 1 to 4, wherein the optical path splitting unit is arranged on the optical path of the laser beam combined by the combining member. 10. A laser light source that emits a plurality of laser beams having different wavelengths, an optical path splitting unit that splits the optical paths of the plurality of laser beams from the laser light source, and each on the optical path split by the optical path splitting unit. A plurality of optical fibers on which the laser beams are respectively incident, and a plurality of optical fibers that are respectively provided between the optical path splitting unit and the plurality of optical fibers and select the wavelength of the laser beam incident on each of the plurality of optical fibers. And a confocal scanning laser microscope optical system and a total reflection fluorescence microscope optical system that use the laser beams that have passed through the plurality of optical fibers, respectively, in a single microscope. And microscope system.