JP2000088751A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent a sample from being vibrated carelessly by preventing an optical axis from deviating due to the move of an XY stage even when light cannot be guided by a fiber. SOLUTION: A laser microscope is provided with an extremely short pulse laser oscillator 21 for outputting a laser beam, an XY stage 22 with an X stage that can travel in an X-axis direction and a Y stage that can travel in a Y-axis direction, a stage 2 for placing the sample for separating from the XY stage 22, a microscope body 5 that is provided with a scanning optical system 11 for scanning the sample on the stage 2 with a laser beam and is fixed and arranged on the XY stage, a mirror 34 that is fixed to follow the move of the Y stage and guides a laser beam to the scanning optical system 11, a mirror that is provided at the Y stage and guides the laser beam to the mirror 34, and a mirror 32 that is provided at the X stage and guides the laser beam to the mirror.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser microscope having improved coherent light introducing means for observing a sample by irradiating the sample with coherent light to cause a two-photon phenomenon.

[0002]

2. Description of the Related Art When a contact operation such as electrophysiology or cell operation is performed on a specimen under a microscope, external vibrations hinder the contact operation and the specimen may be destroyed.
In order to eliminate vibrations from outside the microscope,
The stage on which the specimen is mounted is fixed to the surface plate, and the microscope body is configured to be movable in the horizontal plane direction, for example, to inadvertently apply vibrations to the electrophysiological electrodes and manipulators that have come into contact with the specimen. The field of view can be moved without the need.

FIG. 4 is a known basic configuration diagram of a microscope provided with a stage on which such a sample is mounted and in which the microscope body is mounted on another stage so as to be movable. A stage 2 for a sample is provided on the surface plate 1, and a sample 3 is placed on the stage 2.

An XY stage 4 for the microscope body is provided on the surface plate 1 separately from the stage 2 for the specimen. The XY stage 4 is composed of plates 4a to 4c, of which the plate 4a is fixed on the surface plate 1, the plate 4b moves in the horizontal direction in the drawing, and the plate 4c moves in the direction perpendicular to the drawing. It is moving. On the XY stage 4, an epi-illumination system 6, an objective lens 7, an eyepiece 8, and an imaging optical element 9 are provided as a microscope main body 5.

Accordingly, the microscope main body 5 includes the XY stage 4
Of the objective lens 7 with respect to the stage 2 on which the sample 3 is mounted, so that the position of the objective lens 7 can be moved on a horizontal plane. Note that focusing on the sample 3 is performed by moving the objective lens 7 up and down.

The operation of the microscope having such a configuration will be described. Illumination light passing through the epi-illumination system 6 is focused on the specimen 3 by the objective lens 7. By this illumination, fluorescent light or reflected light is emitted from the specimen 3, and the fluorescent light or reflected light can be observed again as an image of the specimen 3 by the eyepiece 8 or the imaging optical element 9 through the objective lens 7.

At this time, the observation position of the specimen 3 is set at a desired observation region by moving the microscope main body 5 by driving the plates 4b and 4c of the XY stage 4 on which the microscope main body 5 is mounted to XY in the horizontal direction. It can be moved and the focusing on the specimen 3 is performed by moving the objective lens 7 up and down.

When a contact operation such as electrophysiology or cell operation is performed on the specimen 3, for example, an electrode for electrophysiology or a manipulator 10 is brought into contact with the specimen 3.

In such a microscope, the stage (2) on which the sample 3 is mounted is not moved up and down as in a normal microscope, ie, the sample 3 and the manipulator 10 in contact with the sample 3 are not moved. The positioning and focusing of the specimen 3 can be performed without giving careless vibration.

On the other hand, a case where the observation of the specimen 3 is applied to a laser microscope will be described. A scanning optical unit 11 is installed in the microscope main body 5 instead of the imaging optical element 9, and a fiber is connected to the scanning optical unit 11. 12
The laser light source 13 is connected via a flexible material such as.

With such a configuration, the laser light source 13
Is output to the scanning optical unit 11 by the fiber 12, is scanned on the image plane of the objective lens 7, and is condensed on the specimen 3 by the objective lens 7. The fluorescence from the specimen 3 excited by the irradiation of the coherent light is again guided to the scanning optical unit 11 through the objective lens 7 and enters the optical conversion element 31 installed in the scanning optical unit 11. By constructing the output from the optical conversion element 31 as an image, it can be observed as an image of the specimen 3.

The observation position of the specimen 3 is determined by the respective plates 4b, 4b of the XY stage 4 on which the microscope main body 5 is mounted as described above.
By moving the microscope body 5 by driving c horizontally and XY in the horizontal direction, it can be moved to a desired observation site.

During the movement of the observation site, the coherent light output from the laser light source 13
Scanning optical unit 1 guided by a flexible material such as
If the light is guided to 1, the optical axis of the coherent light does not shift due to the driving of the XY stage 4.

[0014]

However, when observing the sample 3 by causing a two-photon phenomenon, an ultrashort pulse laser oscillator is used as a light source. When this ultra-short pulse laser oscillator is applied to the laser light source having the above-described configuration, the ultra-short pulse laser light output from the ultra-short pulse laser oscillator passes through the fiber 12 to increase its pulse width. There is a possibility that the photon phenomenon does not easily occur, and in some cases, the two-photon phenomenon does not occur. For this reason, when the ultrashort pulse laser light for causing the two-photon phenomenon is used, the ultrashort pulse laser light cannot be guided to the scanning optical unit 11 by the fiber 12.

An object of the present invention is to provide a laser microscope which does not cause an optical axis shift due to the movement of the stage even when the light cannot be guided by the fiber and does not give an inadvertent vibration to the sample.

[0016]

According to a first aspect of the present invention, there is provided a laser light source for outputting a laser beam, and an X-ray movable in an X-axis direction.
An XY stage including a stage and a Y stage movable in the Y-axis direction; a sample mounting table for mounting the sample so as to be separated from the XY stage; and a laser beam applied to the sample on the sample mounting table. A microscope body provided with a scanning optical system for scanning and fixedly arranged on an XY stage; a scanning system introducing means fixed to follow the movement of the Y stage and guiding laser light to the scanning optical system; A first optical axis adjusting means for guiding the laser light to the scanning system introducing means, and a second optical axis adjusting means provided on the X stage for guiding the laser light to the first optical axis adjusting means. It is a laser microscope provided.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to the drawings. The same parts as those in FIG. 4 are denoted by the same reference numerals, and detailed description thereof will be omitted. 1 is a plan view of a laser microscope, FIG. 2 is a view from the left side, and FIG. 3 is a front view.

An ultrashort pulse laser oscillator 21, an XY stage 22, and a sample stage (sample mounting table) 2 on which the sample 3 is mounted are provided on the surface plate 20 as light sources.

The ultrashort pulse laser oscillator 21 has a function of outputting an ultrashort pulse laser beam Q having a wavelength for causing a two-photon phenomenon. The XY stage 22 includes a fixed stage 22a, an X stage 22b, and a Y stage 22c.
Are stacked. Among them, fixed stage 22
a is fixed on the surface plate 20. On the fixed stage 22a, the X stage 22b is in the X direction, that is, the same as the optical axis direction of the ultrashort pulse laser beam Q output from the ultrashort pulse laser oscillator 21. It is provided movably in the direction. On the X stage 22b, a Y stage 22c is moved in the Y direction, that is, in the direction perpendicular to the optical axis direction of the ultrashort pulse laser beam Q output from the ultrashort pulse laser oscillator 21 (in the moving direction of the X stage 22b). (Vertically with respect to the vertical direction).

On the XY stage 22, supporting columns 23
The scanning optical unit 11 is provided by
An epi-illumination system 6, an imaging lens 24, an objective lens 7, an eyepiece 8, an imaging optical element 9, and a mirror 25 for guiding ultrashort pulse laser light from the scanning optical unit 11 to the imaging lens 24 are provided as the microscope main body 5. Have been.

The scanning optical unit 11 is provided with dichroic mirrors 26a and 26b and scanning mirrors 27a and 27b for scanning in directions orthogonal to each other, and has a photometric filter 28 on a transmission optical path of the dichroic mirror 26a.
The lens 29, the pinhole 30, and the photoelectric conversion element 31 are arranged.

Therefore, the scanning optical unit 11 reflects the ultrashort pulse laser beam Q from the ultrashort pulse laser oscillator 21 on the dichroic mirrors 26a and 26b, scans the scanning mirrors 27a and 27b, and passes through the relay lens 35 through the microscope 35. The specimen 3 is sent to the main body 5 and
Is transmitted from the scanning mirrors 27b and 27a and the mirror 26b through the dichroic mirror 26a to the photoelectric conversion element 31 through the photometric filter 28, the lens 29, and the pinhole 30.

The X stage 22b of the XY stage 22
, A mirror 32 as a second optical axis adjusting means is fixedly provided, and moves in the same direction (X-axis direction) as the optical axis direction of the ultrashort pulse laser light with the movement of the X stage 22b. ing. The mirror 32 is provided at an angle at which the extremely short pulse laser light Q is reflected in the vertical direction, that is, in the Y-axis direction.

A mirror 33 as a first optical axis adjusting means is fixedly provided on the Y stage 22c, and is moved in a direction perpendicular to the optical axis direction of the ultrashort pulse laser beam Q with the movement of the Y stage 22c. (In the Y-axis direction). The mirror 33 is provided at an angle at which the extremely short pulse laser beam Q reflected by the mirror 32 is reflected in a vertically upward direction, that is, in the Z-axis direction.

Further, the scanning optical unit 11 on the optical path of the ultrashort pulse laser beam Q reflected by the mirror 33 is provided with a fixed mirror 34 as a scanning system introducing means. The mirror 34 is provided at an angle at which the very short pulse laser light reflected by the mirror 33 is reflected and guided to the dichroic mirror 26 a in the scanning optical unit 11.

Next, the operation of the laser microscope configured as described above will be described. The ultrashort pulse laser light Q output from the ultrashort pulse laser oscillator 21 is
The light is reflected by the mirror 3 and the mirror 34 and enters the scanning optical unit 11.

In the scanning optical unit 11, a very short pulse laser beam Q is applied to a dichroic mirror 26a and a mirror 2a.
The laser beam Q is reflected two-dimensionally and scanned two-dimensionally by the scanning mirrors 27 a and 27 b and sent to the microscope main body 5 through the relay lens 35.

In the microscope main body 5, the ultrashort pulse laser beam Q scanned two-dimensionally is reflected by the mirror 25, and converted into a light beam diameter that satisfies the pupil diameter of the objective lens 7 through the imaging lens 24. It is sent to the objective lens 7 and focused on the cross section 3 a of the specimen 3 by the objective lens 7.

The fluorescent light generated in the specimen 3 returns from the objective lens 7 to the scanning optical unit 11 through the imaging lens 24, the mirror 25, and the relay lens 35, and returns to the scanning mirrors 27.
b, 27a, and the mirror 26b are reflected and transmitted through the dichroic mirror 26a. Further, the fluorescence wavelength is selected by the photometric filter 28, an image is formed on the surface of the pinhole 30 by the lens 29, and the light passes through the pinhole 30 and is subjected to photoelectric conversion. Element 3
Measured at 1.

When the observation field of view of the sample 3 is moved, the X stage 22 of the XY stage 22 is moved.
The scanning optical unit 11 and the microscope main body 5 are moved on the XY plane by driving the b stage and the Y stage 22c, respectively.

The X stage 22b moves in the same direction (X-axis direction) as the direction of the optical axis of the ultrashort pulse laser beam Q, and is integrated with the X stage 22b to form a mirror 32.
Also moves in the same direction as the optical axis direction of the ultrashort pulse laser beam Q. Thus, even if the X stage 22b moves, the position where the ultrashort pulse laser beam Q hits the mirror 32 does not change.

The Y stage 22c moves in the direction (Y axis direction) perpendicular to the optical axis direction of the ultrashort pulse laser beam Q, and the mirror 33 is integrated with the Y stage 22c. It moves in a direction perpendicular to the optical axis direction of the short pulse laser light Q. Thus, even if the Y stage 22c moves, the position where the ultrashort pulse laser light Q reflected by the mirror 32 hits the mirror 33 does not change.

Further, the mirror 33, the mirror 34, the scanning optical unit 11 and the microscope body 5 are all
2C, the XY stage 22 moves, that is, the scanning optical unit 11 and the XY plane of the microscope main body 5 for moving the observation visual field, unless the position of the ultrashort pulse laser beam Q hitting the mirror 33 is changed. Irrespective of the upward movement, the optical axis of the ultrashort pulse laser beam Q incident on the scanning optical unit 11 does not shift.

As described above, in one embodiment,
X stage 22b moving in the same direction as the optical axis of ultrashort pulse laser beam Q output from ultrashort pulse laser oscillator 21, and optical axis of ultrashort pulse laser beam Q output from ultrashort pulse laser oscillator 21 Stage 22c that moves in a direction perpendicular to the direction and mounts the scanning optical unit 11 and the microscope main body 5, and the X and Y stages 2
2b and 22c are provided respectively, and are provided with mirrors 32 and 33 for guiding the ultrashort pulse laser light Q output from the light source to the scanning optical unit 11, so that observation of the sample 3 in which light is difficult to guide by a fiber, for example, Even when the sample 3 is observed by irradiating the sample 3 with the short-pulse laser light Q to cause a two-photon phenomenon, the XY stage 22 is driven to move the observation field of the sample 3 and the scanning optical unit 11 and the microscope main body. Even if 5 is moved on the XY plane, the optical axis of the ultrashort pulse laser beam Q incident on the scanning optical unit 11 does not shift regardless of the movement. In addition, the observation field of view can be moved without inadvertently applying vibration to the sample 3, so that the pulse width of the ultrashort pulse laser beam Q is increased, as in the case of using a fiber.
It does not mean that the two-photon phenomenon does not easily occur on the surface, or that the two-photon phenomenon does not occur in some cases.

In the above embodiment, mirrors are used as the first and second optical axis adjusting means and the scanning system introducing means in order to cause the two-photon phenomenon. However, the two-photon phenomenon is caused. If it is not necessary, the invention is not limited to a mirror. For example, a prism can be used.
In addition, each is reflected by one mirror,
An optical path may be deflected by inserting a mirror or an optical element between the mirrors.

[0036]

As described above in detail, according to the present invention, even when the light cannot be guided by the fiber, the laser does not shift the optical axis due to the movement of the XY stage and does not inadvertently vibrate the sample. A microscope can be provided.

[Brief description of the drawings]

FIG. 1 is a plan view showing an embodiment of a laser microscope according to the present invention.

FIG. 2 is a configuration diagram of the laser microscope viewed from a left side surface.

FIG. 3 is a front configuration diagram of the laser microscope.

FIG. 4 is a configuration diagram of a conventional microscope.

[Explanation of symbols]

 2: stage, 3: specimen, 5: microscope body, 6: epi-illumination system, 24: imaging lens, 7: objective lens, 8: eyepiece, 9: imaging optical element, 11: scanning optical unit, 20: fixed Board: 21: ultrashort pulse laser oscillator, 22: XY stage, 22a: fixed stage, 22b: X stage, 22c: Y stage, 32: mirror, 33: mirror, 34: mirror.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G043 AA03 BA16 CA03 DA06 EA01 FA01 FA02 GA02 GB01 GB19 HA01 HA02 KA08 KA09 LA03 2H052 AA08 AA09 AC04 AC07 AC15 AC27 AC34 AD19 AF07 AF14 AF19

Claims (1)

[Claims] 1. An XY stage having a laser light source for outputting laser light, an X stage movable in an X-axis direction and a Y stage movable in a Y-axis direction, and a sample separated from the XY stage. And a microscope main body fixedly arranged on the XY stage, the microscope main body including a scanning optical system for scanning the sample on the sample mounting table with the laser light. A scanning system introducing means fixed to follow the movement for guiding the laser light to the scanning optical system; and a first optical axis provided on the Y stage for guiding the laser light to the scanning system introducing means. A laser microscope comprising: an adjusting unit; and a second optical axis adjusting unit provided on the X stage for guiding the laser beam to the first optical axis adjusting unit.