JP2016070961A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To surely avoid a position other than an observation target portion accompanied by a rotational movement of a measurement head from being inadvertently irradiated with laser light.SOLUTION: A laser microscope 1 is provided that comprises: a stage 6 that loads a sample S; a measurement head 9 that irradiates the sample S on the stage 6 with laser light L emitted from a laser light source 2, and holds an objective optical system 8 converging observation light F occurring in the sample S due to the irradiation of the laser light L; a rotation mechanism 10 that supports the measurement head 9 rotatably around a prescribed rotation axial line B with respect to the stage 6; and laser light prohibition means 13 that prohibits emission of the laser light L from the objective optical system 8 at least when the rotation mechanism 10 causes the measurement head 9 to rotate and move.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a laser microscope.

  2. Description of the Related Art Conventionally, there is known a laser microscope in which a measurement head that holds an objective lens is provided so as to be rotatable about a horizontal rotation axis (see, for example, Patent Document 1). According to such a laser microscope, the direction of the optical axis of the objective lens with respect to the specimen on the stage changes due to the rotational movement of the measuring head, so that the specimen can be observed not only in the vertical direction but also in various directions. . For example, in the field of brain science, the brain in a living body can be observed from a horizontal direction or an oblique direction.

Japanese Patent No. 4681834

  However, in the laser microscope as disclosed in Patent Document 1, the laser beam emitted from the objective lens moves with the rotational movement of the measurement head. Therefore, there is a possibility that the laser light is inadvertently irradiated at a position other than the observation part of the specimen when the measuring head is rotated. When the specimen is a biological sample, it is easily affected by, for example, fading of fluorescence due to carelessly irradiated high energy laser light.

  The present invention has been made in view of the above-described circumstances, and can reliably avoid inadvertently irradiating a laser beam to a position other than the observation target site with the rotational movement of the measurement head. An object of the present invention is to provide a laser microscope that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a stage on which a specimen is placed, and objective optics that irradiates the specimen on the stage with laser light emitted from a laser light source and collects observation light generated on the specimen by the irradiation of the laser light. A measurement head that holds the system, a rotation mechanism that rotates the measurement head around a horizontal rotation axis, and at least the laser from the objective optical system when the measurement head is rotationally moved by the rotation mechanism Provided is a laser microscope comprising laser light prohibiting means for prohibiting light emission.

  According to the present invention, an observation light image of a specimen is acquired by focusing observation light generated by irradiating the specimen with laser light from the objective optical system by the objective optical system. Here, the direction of the optical axis of the objective optical system with respect to the specimen on the stage can be changed by rotating the measuring head around the rotational axis by the rotation mechanism, so that the observation light image obtained by observing the specimen from different directions Can be obtained.

  In this case, while the measuring head is rotating, the laser beam prohibiting means forcibly prohibits the emission of the laser beam from the objective optical system. Therefore, it is possible to reliably avoid inadvertently irradiating the laser beam to a position other than the observation target site with the rotational movement of the measurement head.

In the above-mentioned invention, it comprises a rotation detecting means for detecting the rotational movement of the measuring head around the rotational axis, and the laser beam prohibiting means is used when the rotational movement of the measuring head is detected by the rotational detecting means. The emission of the laser beam may be prohibited.
By doing in this way, when the user starts the rotational movement of the measuring head while the laser beam is emitted from the objective optical system, the rotational movement of the measuring head is detected by the rotation detecting means, and the objective optical system The laser beam emission from the laser beam is forcibly stopped by the laser beam prohibiting means. Thereby, the emission of the laser beam from the objective optical system can be stopped substantially simultaneously with the start of the rotational movement of the measuring head.

Further, in the above invention, the laser light prohibiting means includes a permission state that permits the emission of the laser light from the objective optical system and a prohibition state that prohibits the emission of the laser light from the objective optical system. There may be provided a rotation permission means that is provided so as to be switchable by a user and that permits the rotation movement of the measurement head by the rotation mechanism only when the prohibition state is selected by the laser light prohibition means. .
By doing so, the user cannot rotate the measurement head unless the user performs a switching operation of the laser beam prohibiting means to the prohibited state. Therefore, it is possible to reliably avoid the user from rotating the measuring head while the laser beam is emitted from the objective optical system.

Further, in the above-mentioned invention, there is provided brake means for fixing the measurement head at a constant rotation angle around the rotation axis, and the laser beam prohibiting means is when the measurement head is not fixed by the brake means. In addition, the emission of the laser beam may be prohibited.
In this way, when the measuring head is in a state in which the measuring head can be rotated by the rotating mechanism, the laser beam prohibiting means removes the objective optical system from the objective optical system regardless of whether the measuring head is rotating or stationary. The laser beam emission is forcibly prohibited. Therefore, inadvertent irradiation of the laser beam to a position other than the observation target part of the specimen can be avoided more reliably.

Further, in the above invention, a fine rotation mechanism for rotating the measurement head about the rotation axis with an angle determination accuracy higher than that of the rotation mechanism is provided, and the laser beam prohibiting means is configured to measure the measurement by the fine rotation mechanism. When the head is rotating, emission of the laser light from the objective optical system may be permitted.
In this way, the laser beam is prohibited from being irradiated on the sample S during the coarse adjustment of the observation position and the observation direction by the rotation mechanism, and the laser beam is actually emitted during the fine adjustment of the observation position and the observation direction by the fine rotation mechanism. It is possible to set the observation position and the observation direction with high accuracy while irradiating the sample.

In the above invention, a low output light illumination system for irradiating the sample from the objective optical system with low output light having a wavelength in the visible range may be provided independently of the laser light prohibiting means. .
During the rotational movement of the measuring head, irradiation of the laser beam to the specimen is prohibited, so that it is possible to confirm the exact irradiation position and irradiation direction (that is, the observation position and observation direction) of the laser light on the specimen. Can not. On the other hand, the low-power illumination system can irradiate the sample with laser light even while the measuring head is rotating, so the low-power light can be used to accurately determine the irradiation position and direction of the laser light. And it can be adjusted easily.

  According to the present invention, there is an effect that it is possible to reliably avoid inadvertently irradiating the laser beam to a position other than the observation target site with the rotational movement of the measurement head.

1A is a front view and FIG. 1B is a side view illustrating an overall configuration of a laser microscope according to an embodiment of the present invention. It is a front view explaining the rotational movement of the measuring head by the rotation mechanism of the laser microscope of FIG. It is a flowchart which shows the usage method of the laser microscope of FIG. It is the (a) front view and the (b) side view which show the whole structure of the modification of the laser microscope of FIG. FIG. 5 is a front view for explaining the rotational movement of the measuring head by the fine rotation mechanism of the laser microscope of FIG. 4.

Below, laser microscope 1 concerning one embodiment of the present invention is explained with reference to drawings.
As shown in FIGS. 1A and 1B, the laser microscope 1 according to the present embodiment includes a laser light source 2 and a microscope main body that scans a sample S with a laser light L incident from the laser light source 2. 3 and a photodetector 4 that detects fluorescence (observation light) F generated in the specimen S by irradiation of the laser light L.

The specimen S is a biological sample such as a small animal such as a mouse or a cell.
The microscope main body 3 includes a base 5, a stage 6 fixed on the base 5, a measurement head 9 that holds the scanning optical system 7 and the objective optical system 8, and the measurement head 9 around the rotation axis B in the horizontal direction. And a brake (brake means) 11 for preventing the rotational movement of the measuring head 9 by the rotation mechanism 10.
The stage 6 has a substantially horizontal placement surface 6a on which the specimen S is placed.

The measuring head 9 holds the scanning optical system 7 and the objective optical system 8, and includes a housing 12 connected to the laser light source 2 and the photodetector 4 through the optical fibers 21 and 22, the objective optical system 8, and the scanning optical system. 7 and a shutter (laser light prohibiting means) 13 disposed between the two.
The scanning optical system 7 includes, for example, a pair of galvanometer mirrors, and scans the laser light L incident into the housing 12 from the laser light source 2 via the optical fiber 21 in the biaxial direction intersecting the optical axis of the laser light L. However, the light is reflected toward the objective optical system 8.
The objective optical system 8 irradiates the sample S on the stage 6 with the laser light L incident from the scanning optical system 7 and condenses the fluorescence F emitted from the sample S.

  The shutter 13 prohibits the open state (permitted state) in which the laser light L is allowed to pass from the scanning optical system 7 toward the objective optical system 8 and the passage of the laser light L from the scanning optical system 7 toward the objective optical system 8. A closed state (prohibited state) can be alternatively selected. Switching between the open state and the closed state of the shutter 13 is performed by manual switching by a user operation and automatic switching based on a permission signal received from the brake 11 as described later. Specifically, when receiving the permission signal from the brake 11, the shutter 13 switches between an open state and a closed state according to a user operation. On the other hand, the shutter 13 always selects the closed state when the permission signal is not received, and rejects the switching operation to the open state by the user.

  Reference numeral 14 is disposed between the scanning optical system 7 and the objective optical system 8, transmits the laser light L traveling from the scanning optical system 7 toward the objective optical system 8, and passes from the objective optical system 8 to the scanning optical system 7. It is a dichroic mirror that reflects the fluorescent light F traveling toward it. The fluorescence F reflected by the dichroic mirror 14 is guided to the photodetector 4 by the optical fiber 22 and detected by the photodetector 4.

The rotating mechanism 10 includes a columnar shaft 15 extending in the horizontal direction and an arm 16 extending in a vertical plane.
The shaft 15 is fixed with respect to the base 5.
The arm 16 supports the measuring head 9 at one end so that the optical axis A of the objective optical system 8 is directed toward the other end. Between the arm 16 and the measurement head 9, there is provided a moving mechanism 17 that moves the measurement head 9 in three directions of X, Y, and Z. The Z direction is the direction of the optical axis A of the objective optical system 8, and the X direction and the Y direction are directions that intersect the optical axis A of the objective optical system 8.

  The arm 16 has a cylindrical sleeve 16 a in which a through hole in a direction perpendicular to the longitudinal axis of the arm 16 is formed at the other end. The sleeve 16 a is fitted on the outer side of the shaft 15 so as to be rotatable around a rotation axis B that is a central axis of the shaft 15. By rotating the sleeve 16a around the rotation axis B with respect to the fixed shaft 15, as shown in FIG. 2, the measurement head 9 of the objective optical system 8 aligned with the sample S on the mounting surface 6a is obtained. The optical axis A of the objective optical system 8 with respect to the sample S is changed by rotating around the rotation axis B passing through the focal position or the vicinity thereof. The rotational movement of the measuring head 9 may be performed manually by the user or electrically by a motor (not shown).

  The brake 11 includes, for example, a brake pad (not shown) made of an elastic member such as rubber. By pressing the brake pad against the outer peripheral surface of the sleeve 16a, the brake 11 is prevented from rotating around the rotation axis B of the sleeve 16a. A braking force is generated by friction between the pad and the sleeve 16a, and the rotation of the sleeve 16a around the rotation axis B is prevented by the braking force.

  The brake 11 includes a means for detecting the magnitude of the braking force applied to the sleeve 16a by the brake 11, for example, a load sensor for detecting a load acting on the brake pad. The brake 11 transmits a permission signal only to the shutter 13 when the braking force applied to the sleeve 16a is equal to or greater than a predetermined threshold, and does not transmit a permission signal to the shutter 13 when the braking force is less than the predetermined threshold. That is, only when the arm 16 is stably fixed by the brake 11, the shutter 13 can be switched to the open state, and the laser light L can be emitted from the objective optical system 8.

Further, the laser microscope 1 includes a low output light illumination system 18 that irradiates the sample S with the low output light L ′.
The low output light illumination system 18 includes a light source 19 that outputs a low output light L ′, and an optical axis A of the objective optical system 8 that converts the low output light L ′ incident from the light source 19 through the optical fiber 23 into the housing 12. And a dichroic mirror 20 that reflects toward the objective optical system 8.

The low output light L ′ has a wavelength in the visible range so that the user can observe with the naked eye. Further, the low output light L ′ has energy lower than that of the laser light L that does not affect the sample S.
The dichroic mirror 20 is disposed on the optical axis A of the objective optical system 8 between the shutter 13 and the objective optical system 8, and emits low output light L ′ at a position closer to the objective optical system 8 than the shutter 13. The laser beam L is combined with the optical path. Thereby, the low output light L ′ can be emitted from the objective optical system 8 at an arbitrary timing independently of the open / closed state of the shutter 13. The dichroic mirror 20 reflects the low output light L ′ and transmits the laser light L and the fluorescence F.

Next, the operation of the laser microscope 1 configured as described above will be described.
In order to observe the specimen S with the laser microscope 1 according to the present embodiment, first, as shown in FIG. 3, the specimen S is placed on the placement surface 6a of the stage 6 (step S1). Next, the observation position and the observation direction of the sample S are adjusted while performing preliminary observation using the low output light L ′. Specifically, the brake 11 is released (step S2), and irradiation of the low output light L ′ from the objective optical system 8 to the sample S is started (step S3). In the preliminary observation in step S3, the user irradiates the low output light L ′ irradiated on the specimen S to the desired observation position of the specimen S from the desired observation direction while visually confirming the low output light L ′ irradiated on the specimen S. In this manner, the rotation angle of the measurement head 9 around the rotation axis B is adjusted (step S4), and the measurement head 9 is fixed by the brake 11 (step S5).

The low output light L ′ and the laser light L are irradiated to the same position of the sample S from the same direction through the same optical path. Therefore, the observation position and the observation direction by the laser light L can be set in a desired state in advance without affecting the sample S by using the low output light L ′.
Next, the low output light L ′ is stopped, the output of the laser light L from the laser light source 2 is started, and the shutter 13 is switched to the open state to start the main observation with the laser light L (step S6).

  The laser light L output from the laser light source 2 is guided to the scanning optical system 7 in the measurement head 9 via the optical fiber 21 and is two-dimensionally scanned by the scanning optical system 7 and then passed through the objective optical system 8. The sample S is irradiated. The fluorescence F generated when the laser light L is two-dimensionally scanned at the observation position of the specimen S is collected by the objective optical system 8 and guided to the photodetector 4 through the dichroic mirror 14 and the optical fiber 22. The The intensity information of the fluorescence F detected by the photodetector 4 is correlated with the scanning position of the laser light L by an image processor (not shown) to form a two-dimensional image.

  When it is desired to observe another observation position of the specimen S after completion of the main observation or to observe the specimen S from another observation direction (NO in step S7), the objective optical is switched by switching the shutter 13 to the closed state. The emission of the laser beam L from the system 8 is stopped (step S8), and then steps S2 to S6 are repeated.

In this case, even if the user neglects the operation of stopping the laser light L in step S8 and releases the brake 11 while the laser light L is irradiated from the objective optical system 8 to the specimen S, the brake 11 When the shutter 13 is automatically closed in response to the release of the laser beam L, the laser beam L immediately disappears.
In addition, during the main observation using the laser beam L, even when the brake 11 is loosened for some reason and the measurement head 9 is not fixed, the shutter 13 responds to the loosening of the brake 11. By automatically closing, the laser beam L immediately disappears.

  As described above, when the measuring head 9 is in a state of being able to rotate and move while the sample S is being irradiated with the laser light L, the emission of the laser light L from the objective optical system 8 is forcibly stopped immediately. The laser light L is not accidentally irradiated to a position other than the desired observation position. The high-energy laser light L such as the pulsed laser light used for the main observation of the specimen S in the laser microscope 1 easily interacts with the biological sample, and easily affects the biological sample such as fading of the fluorescent material. According to the present embodiment, the biological sample that is the specimen S can be protected from such inadvertent irradiation of the high-energy laser beam L.

In the present embodiment, in addition to the rotation mechanism 10, as shown in FIGS. 4A and 4B, a fine rotation mechanism 24 may be provided.
Similar to the rotation mechanism 10, the fine rotation mechanism 24 includes a fine movement arm 25 provided with a sleeve 25 a at the end, and the measurement head 9 is supported by the fine movement arm 25.

  The fine movement arm 25 is arranged in parallel with the arm 16, and the sleeve 25a of the fine movement arm 25 is rotatably fitted to the outside of the sleeve 16a. Thereby, as shown in FIG. 5, the fine movement arm 25 can be rotated around the rotation axis B with respect to the arm 16 independently of the brake 11. Here, the fine movement arm 25 can be rotationally moved by a predetermined minute angle with respect to the arm 16 by a so-called click mechanism or the like so that the rotation angle around the rotation axis B can be determined with higher accuracy than the arm 16. Yes.

  In this way, after performing coarse adjustment of the observation position and the observation direction by the rotational movement of the arm 16 using the low output light L ′, the sample S is actually irradiated with the laser light L while the fine movement arm 25 is moved. Fine adjustment of the observation position and the observation direction by rotational movement can be performed.

  In the present embodiment, the shutter 13 is automatically closed when the braking force applied to the sleeve 16a by the brake 11 is less than a predetermined threshold value, but instead, the rotation around the rotation axis B of the arm 16 is performed. Rotation detecting means (not shown) such as an encoder for detecting the rotational movement of the arm 16 is provided, and the shutter 13 is automatically closed when the rotational movement of the arm 16 is detected by the rotation detecting means. Also good.

  In this way, when the user starts the rotational movement of the measuring head 9 while the laser beam L is emitted from the objective optical system 8, the rotation detecting means immediately detects the start of the rotational movement of the measuring head 9. Thus, the shutter 13 is closed. Even in this way, the specimen S can be protected from inadvertent irradiation of the laser light L.

Further, in this embodiment, the brake (rotation permission means) 11 acquires a signal indicating the open / closed state of the shutter 13 from the shutter 13, and the arm 16 is released only when the shutter 13 is in the closed state. It may be configured to be possible.
By doing in this way, the user cannot release the brake 11 unless the user performs a switching operation of the shutter 13 to the closed state. Even in this case, the specimen S can be reliably protected from inadvertent irradiation of the laser light L.

  In the present embodiment, the shutter 13 disposed in the optical path between the laser light source 2 and the objective optical system 8 is used as the laser light prohibiting means. Instead, for example, the laser light source 2 is used. The emission of the laser light L from the objective optical system 8 may be forcibly stopped using other means such as turning off the light.

DESCRIPTION OF SYMBOLS 1 Laser microscope 2 Laser light source 3 Microscope main body 4 Photo detector 5 Base 6 Stage 6a Mounting surface 7 Scanning optical system 8 Objective optical system 9 Measuring head 10 Rotating mechanism 11 Brake (brake means, rotation permission means)
12 Housing 13 Shutter (Laser light prohibition means)
14, 20 Dichroic mirror 15 Shaft 16 Arm 16a, 25a Sleeve 17 Movement mechanism 18 Low power illumination system 19 Light source 21, 22, 23 Optical fiber 24 Fine rotation mechanism 25 Fine movement arm S Sample L Laser light F Fluorescence (observation light)
L 'Low power light

Claims (6)

A stage for placing the specimen;
A measurement head that holds an objective optical system that irradiates the specimen on the stage with laser light emitted from a laser light source and collects observation light generated in the specimen by the irradiation of the laser light;
A rotation mechanism for rotating the measuring head around a horizontal rotation axis;
A laser microscope comprising at least laser light prohibiting means for prohibiting emission of the laser light from the objective optical system when the measuring head is rotationally moved by the rotating mechanism. A rotation detecting means for detecting a rotational movement of the measuring head around the rotation axis;
2. The laser microscope according to claim 1, wherein the laser beam prohibiting unit prohibits the emission of the laser beam when the rotation detecting unit detects the rotational movement of the measuring head. The laser light prohibiting means can be switched by a user between a permission state in which the emission of the laser light from the objective optical system is permitted and a prohibition state in which the emission of the laser light from the objective optical system is prohibited. Provided,
2. The laser microscope according to claim 1, further comprising a rotation permission unit that permits the rotation movement of the measuring head by the rotation mechanism only when the prohibited state is selected by the laser beam prohibiting unit. Brake means for fixing the measurement head at a constant rotation angle around the rotation axis,
The laser microscope according to any one of claims 1 to 3, wherein the laser beam prohibiting unit prohibits emission of the laser beam when the measurement head is not fixed by the brake unit. A fine rotation mechanism for rotating the measurement head around the rotation axis with a higher angle determination accuracy than the rotation mechanism;
5. The laser light prohibiting means permits the emission of the laser light from the objective optical system when the measuring head is rotationally moved by the fine rotation mechanism. 6. Laser microscope.   The low-output light illumination system that irradiates the sample with low-output light having a visible wavelength from the objective optical system independently of the laser light prohibiting unit. The laser microscope described.

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