JP5771371B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope.

  Conventionally, a resonant galvanometer mirror that scans light from a laser light source in a predetermined X direction and a piezo element that scans light from the light source in a Z direction are provided in two dimensions in a three-dimensional sample. A laser scanning microscope that scans automatically is known (for example, see Patent Document 1).

  The above laser scanning microscope is designed to scan a high-speed resonant scanner (in the X direction) until scanning of the low-speed piezo element (scan in the Z direction) is completed for the purpose of reducing damage to a specimen such as a biological sample. (Scanning) is repeated, and the signals obtained during that time are integrated, and the integrated value is controlled to exceed a predetermined value.

JP 2001-108904 A

  However, according to the laser scanning microscope described above, the sample image is generated by integrating the plurality of scanning lines after setting the light intensity from the laser light source low, so that the resolution of the sample image is reduced. There is an inconvenience.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a laser scanning microscope that can improve the resolution of a specimen image while reducing damage to the specimen.

In order to achieve the above object, the present invention employs the following means.
The present invention includes a laser light source that emits laser light, a first scanning unit that has at least one resonant galvanometer mirror, and scans the sample with the laser light from the laser light source to generate an observation image; A second scanning unit that changes a focal position of a laser beam in an optical axis direction with respect to the sample so as to change an observation depth of the sample; an emission timing of the laser light source; the first scanning unit; and the second scanning unit. A control unit that controls the scanning timing of the first scanning unit , and the control unit uses a synchronization signal of the first scanning unit that is turned on within an effective scanning range for each scanning of each line of the first scanning unit. determined by the effective timing and synchronization signals of the second scanning section observation depth is changed by the second scanning unit is turned on every time reaching the focal position to be subjected to laser scanning according to the first scanning portion determined That when both effective timing of the effective timing, said by repeating the control to emit a laser beam from the laser light source, employing a laser scanning microscope which performs two-dimensional scanning in a plane along the optical axis of the laser beam.

  According to the present invention, the laser light from the laser light source is scanned on the sample by the first scanning unit having at least one resonant galvanometer mirror, and the observation parameter of the sample is changed by the second scanning unit. . In this case, the control unit allows the laser beam to be emitted from the laser light source at the effective timing of both the first scanning unit and the second scanning unit, that is, at the timing when image acquisition is to be performed. This prevents the sample from being irradiated with laser light that is emitted when the scanning unit is not at an effective timing, that is, laser light that does not contribute to the generation of the sample image, and reduces damage to the sample by the laser light. can do. In addition, a sample image can be generated without performing integration of a plurality of scanning lines as in the prior art, and the resolution of the sample image can be improved. Here, the specimen observation parameters are, for example, the specimen observation position and the observation wavelength. The first scanning unit includes at least one resonant galvanometer mirror that scans the laser beam on the observation surface of the specimen, and XY scanning is enabled by combining scanning means such as a galvanometer mirror that scans in a direction orthogonal to the first galvanometer mirror. It may be a thing.

In the above invention, the second scanning unit, Ru changing the focal position of the optical axis of the laser beam relative to the specimen.
By doing in this way, when the scanning direction by the first scanning unit is the X direction, the focal position in the optical axis direction (Z direction) of the laser beam with respect to the sample is changed by the second scanning unit, A two-dimensional image (XZ image) of the specimen can be generated. In this case, the laser beam is emitted from the laser light source at the effective timing of both the first scanning unit and the second scanning unit, thereby preventing the sample from being irradiated with laser light that does not contribute to the generation of the sample image. It is possible to reduce damage to the specimen due to laser light. In addition, when the first scanning unit is configured to be capable of two-dimensional (XY) scanning, the focal position of the laser beam with respect to the specimen in the optical axis direction (Z direction) is changed accordingly. A three-dimensional image (XYZ image) can be generated. In this case, the laser beam is emitted from the laser light source at the effective timing of both the first scanning unit and the second scanning unit, thereby preventing the sample from being irradiated with laser light that does not contribute to the generation of the sample image. It is possible to reduce damage to the specimen due to laser light.

In the reference example of the invention described above, the second scanning unit may relatively move the sample in a direction orthogonal to the optical axis of the laser light.
By doing in this way, even when the desired observation position exceeds the scanning range (observable range) of the first scanning unit, the second scanning unit (for example, the XY stage on which the sample is placed) is located at the sample position. By moving the lens relative to the optical axis, a desired observation position can be observed.

In the above invention, the laser light source may switch the wavelength of the emitted laser light.
By doing so, it is possible to switch the wavelength of the laser light and irradiate the sample, and generate a sample image for each wavelength. In this case, the laser light can be emitted from the laser light source at the effective timing of both the first scanning unit and the second scanning unit. Thereby, it is possible to prevent the sample from being irradiated with laser light that does not contribute to generation of the sample image, and it is possible to reduce damage to the sample by the laser light.

In the above invention, at least one effective timing of the first scanning unit may be included in the effective timing of the second scanning unit.
By doing so, it is possible to reliably irradiate the sample with the laser beam during the effective timing of the second scanning unit and perform scanning by the first scanning unit, and to improve the resolution of the sample image. it can.

  According to the present invention, it is possible to improve the resolution of a specimen image while reducing damage to the specimen.

1 is a schematic configuration diagram of a laser scanning microscope according to an embodiment of the present invention. 2 is a timing chart when the Z stage of FIG. 1 is operated continuously. 3 is a flowchart illustrating processing executed by each unit in the case of FIG. 2. 2 is a timing chart when the Z stage of FIG. 1 is intermittently operated and the wavelength of laser light is switched. 5 is a flowchart illustrating processing executed by each unit in the case of FIG. 4.

A laser scanning microscope 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the laser scanning microscope 1 according to the present embodiment includes a light source unit 10 that emits laser light, a Z stage 27 on which a sample (specimen) A is placed, and a laser from the light source unit 10. An optical unit 20 that irradiates light on the sample A and detects light from the sample A, a control unit 30 that controls each unit and generates an image of the sample A, and a display device 31 that displays an image of the sample A And an input unit 32 for inputting an instruction to the control unit 30.

  The light source unit 10 includes laser light sources 11, 12, and 13 that emit laser light, laser light control units 14, 15, and 16 that control light from the laser light sources 11, 12, and 13, and a laser light control unit, respectively. And an optical coupler 17 for synthesizing the laser beams from 14, 15, and 16.

  The laser light sources 11, 12, and 13 emit, for example, excitation light (laser light) that excites a fluorescent substance in the sample A to generate fluorescence, and emits laser light having different wavelengths. It has become.

  The laser dimmers 14, 15, and 16 are, for example, acousto-optic elements and electro-optic elements. Are modulated or turned ON / OFF, respectively.

  The optical coupler 17 is, for example, a dichroic mirror, and transmits the laser light from the laser light source 11, while reflecting the laser light from the laser light sources 12, 13, thereby causing the laser light from the laser light sources 11, 12, 13. Are designed to be coaxial.

The above dichroic mirror transmits the laser light from the laser light sources 11 and 12 while reflecting the laser light from the laser light source 13 to synthesize the laser light from the laser light sources 11, 12 and 13 coaxially. It is good to do.
Alternatively, the optical coupler 17 has two dichroic mirrors, and the first dichroic mirror transmits the laser light from the laser light source 11, while reflecting the laser light from the laser light source 12, and the second dichroic mirror. The mirror may reflect the laser light from the laser light source 13 while transmitting the laser light from the first dichroic mirror.

  The optical unit 20 condenses the laser light scanned by the XY scanner 22 on the sample A and an XY scanner (first scanning unit) 22 that scans the laser light from the optical coupler 17 on the sample A. An objective lens (not shown) that collects the light from A, a spectroscopic unit 21 that splits the laser light from the optical coupler 17 and the light from the sample A, and the light from the sample A that is split by the spectroscopic unit 21 And a detection unit 25 for detection.

  The XY scanner 22 scans the laser beam from the optical coupler 17 on the sample A in the X direction (one direction on the sample A) and the laser beam from the optical coupler 17 on the sample A. And a Y galvanometer mirror 24 that scans in the Y direction (a direction perpendicular to the X direction on the sample A).

The X galvanometer mirror 23 is a resonant galvanometer mirror such as a resonant galvanometer mirror, for example, and scans the laser beam from the optical coupler 17 on the sample A at high speed and continuously in the X direction.
The Y galvanometer mirror 24 is a normal galvanometer mirror, and scans the laser beam from the optical coupler 17 in the Y direction on the sample A.

The XY scanner 22 operates the X galvanometer mirror 23 and the Y galvanometer mirror 24 in cooperation with each other so that the laser beam synthesized by the optical coupler 17 is two-dimensionally scanned on the sample A.
The X drive timing signal of the X galvanometer mirror 23 is output to the control unit 30.

  The Z stage (second scanning unit) 27 moves in the Z direction (direction orthogonal to the X direction and the Y direction) in a state where the sample A is placed based on a signal (Z drive signal) from the control unit 30. It is supposed to be. Thereby, the focal position of the objective lens in the sample A is changed in the Z direction by moving the Z stage 27 in the Z direction.

  The input unit 32 is an input device such as a keyboard, a mouse, and a touch panel screen, for example, and the selection information is input to the control unit 30 by the user selecting the observation position of the sample A and the laser beam to be used. ing.

The control unit 30 generates a three-dimensional image of the sample A based on the light from the sample A detected by the detection unit 25, the scanning position on the sample A of the XY scanner 22, and the position of the Z stage 27. It is like that.
The control unit 30 controls the emission timing of the laser light sources 11, 12, 13, the scanning timing of the XY scanner 22, and the driving timing of the Z stage 27.

  Specifically, as shown in the timing chart of FIG. 2, the X drive timing signal is turned ON when the X galvanometer mirror 23 scans in the positive direction. Further, in the scanning range (X scanning range) of the X galvanometer mirror 23, the horizontal synchronization signal is turned ON within the effective scanning range used for image generation. The Z drive signal is turned ON while the Z stage 27 is being driven, and the Z synchronization signal is turned ON when the focal position reaches the depth to be observed. Then, in a state where all of the X drive timing signal, the horizontal synchronization signal, and the Z synchronization signal are ON, the laser beam emission timing signal for emitting the laser beam is turned ON.

  By performing signal processing as described above and controlling each unit, the control unit 30 emits laser light from the laser light sources 11, 12, and 13 at the effective timing of both the XY scanner 22 and the Z stage 27. It has become. Here, the valid timing of both the XY scanner 22 and the Z stage 27 is a timing at which the horizontal synchronization signal and the Z synchronization signal are turned on, that is, a timing at which image acquisition is to be performed.

The operation of the laser scanning microscope 1 having the above configuration will be described.
First, the case where the Z stage 27 is continuously operated (moved at a constant speed) will be described below according to the timing chart of FIG. 2 and the flowchart of FIG. Here, the sample A is irradiated with a single laser beam. In this case, based on the laser light emission timing signal from the control unit 30, the laser light may be emitted from all of the laser light sources 11, 12, and 13 to irradiate the sample A with the combined light. , 13 may be irradiated with the laser beam from the at least one of the samples A and 13.

  First, the moving speed (Z speed) of the Z stage 27 is set from the depth interval (observation depth interval) of the sample A to be observed and the scanning time (X scanning time) by the X galvanometer mirror 23 (step S1). . Here, the Z speed is determined in order to match the observation timing with the X scanning time. Thereby, the sample A can be observed with the set observation depth.

Next, a Z drive signal is output in accordance with the X drive timing signal from the X galvanometer mirror 23, and the drive of the Z stage 27 is started (step S2).
Next, whether or not the set observation depth has been reached is determined based on the set Z speed and time (observation depth time) (step S3).
At this time, it may be determined that the set observation depth is reached based on a pulse signal or feedback signal corresponding to the observation depth.

In step S3, when the observation depth is reached, the Z synchronization signal is turned ON (step S4).
Next, in a state where the X drive timing signal is ON and the Z synchronization signal is ON, the laser light emission timing signal is turned ON in accordance with the timing when the horizontal synchronization signal is turned ON, and the laser dimmers 14, 15, 16 are turned on. The laser light is emitted from the light source unit 10 by operating (step S5).

  Further, the laser beam emission timing signal is turned off in accordance with the timing when the horizontal synchronization signal is turned off, and the emission of the laser beam from the light source unit 10 is stopped (step S6).

Next, when the observation depth range is passed, the Z synchronization signal is turned OFF (step S7).
Next, it is determined whether or not the observation to all the observation depths has been completed (step S8). If the observation has not been completed, the processes from step S3 to S8 are repeated. 27 is stopped (step S9).

  Hereinafter, the case where the Z stage 27 is stopped (intermittently moved) for each observation depth and the laser beam to be used is switched will be described with reference to the timing chart of FIG. 4 and the flowchart of FIG. In this case, based on the laser beam emission timing signal from the control unit 30, the laser beam is emitted from any one of the laser light sources 11, 12, and 13 to irradiate the sample A.

First, the observation position in the Z direction of the sample A and the laser beam to be used are set (step S11).
Next, the sample A is moved to the observation position set by driving the Z stage 27 (step S12).

Next, the Z synchronization signal is turned ON in accordance with the X drive timing signal from the X galvanometer mirror 23 (step S13).
Next, the laser beam emission timing signal is turned ON in accordance with the timing when the horizontal synchronization signal is turned ON, and the sample A is irradiated with the laser beam (step S14). At this time, the sample A is irradiated with laser light corresponding to the selector signal by switching the selector signal. Specifically, in the example shown in FIG. 4, when the selector signal 1 is turned ON, the laser light emission timing signal 1 is turned ON at the timing when the horizontal synchronization signal is turned ON, and the laser light from the laser light source 11 is turned on. Sample A is irradiated. Further, by turning off the selector signal 1 and turning on the selector signal 2, the laser light emission timing signal 2 is turned on at the timing when the horizontal synchronization signal is turned on, and the laser light from the laser light source 12 is applied to the sample A. Irradiate.

Next, in synchronization with the timing at which the horizontal synchronization signal is turned off, the laser beam is interrupted and the irradiation to the sample A is stopped (step S15).
Next, it is determined whether or not another laser beam is to be used (step S16). If another laser beam is not used, the Z synchronization signal is turned off (step S17).
On the other hand, when another laser beam is used, the laser beam is switched and the processes in steps S14 to S15 are repeated. Specifically, as shown in FIG. 4, by turning ON / OFF the selector signals 1 and 2, the laser beam emission timing signals 1, 2, and 3 are sequentially turned ON at the timing when the horizontal synchronization signal is turned ON. Laser light from each laser light source is sequentially emitted.

  Next, it is determined whether or not the observations at all the observation positions have been completed (step S18). If the observation has not been completed, the processing from steps S12 to S18 is repeated. Observation ends (step S19).

  As described above, according to the laser scanning microscope 1 according to the present embodiment, the laser light from the light source unit 10 is scanned on the sample A by the XY scanner 22 and the observation parameter of the sample A by the Z stage 27. Is changed. Here, the observation parameters are defined by the observation wavelength set according to the wavelength of the selected laser beam, the observation position in the optical axis direction defined by the movement of the Z stage 27, and the scanning position of the XY scanner 22. Observation position in a direction orthogonal to the optical axis.

  In this case, the control unit 30 can emit laser light from the light source unit 10 at the effective timing of both the XY scanner 22 and the Z stage 27. Here, the valid timing of both the XY scanner 22 and the Z stage 27 is the timing when the horizontal synchronization signal and the Z synchronization signal are turned on, that is, the timing at which image acquisition should be performed, as described above. Thereby, it is possible to prevent the sample A from being irradiated with laser light emitted when the XY scanner 22 and the Z stage 27 are not in effective timing, that is, laser light that does not contribute to the generation of the image of the sample A. Damage to the sample A due to the above can be reduced. Further, unlike the conventional technique, an image of the sample A can be generated without performing integration of a plurality of scanning lines, and the resolution of the image of the sample A can be improved.

Further, the Z stage 27 can generate a two-dimensional image (XZ image) of the sample A by changing the focal position of the laser beam with respect to the sample A in the optical axis direction. Further, by performing two-dimensional scanning of the sample A with the XY scanner 22, a two-dimensional image (XY image) of the sample A can be generated. Further, by operating the Z stage 27 together with the two-dimensional scanning by the XY scanner 22, a three-dimensional image (XYZ image) of the sample A can be generated.
In addition, by switching the wavelength of the laser light by the light source unit 10 and irradiating the sample A, an image of the sample A for each wavelength can be generated.

Further, as the second scanning unit, an XY stage that moves the sample A in the direction orthogonal to the optical axis of the laser beam, that is, the XY direction, may be employed instead of the Z stage 27.
In this case, when the place to be observed is outside the scanning range of the XY scanner 22, the XY stage is moved to the observation place, the XY synchronization signal is turned ON in accordance with the X drive timing signal after the XY stage is stopped, The laser beam is turned on in accordance with the horizontal synchronization signal being turned on. Then, the laser beam and the XY synchronization signal are turned OFF in accordance with the OFF of the horizontal synchronization signal, and the next observation place is moved. A two-dimensional image (XY image) of the sample A can be generated by repeating the above operation for the necessary observation locations. In addition to the above XY stage, the three-dimensional image (XYZ image) of the sample A can be generated by including the Z stage 27 of the present embodiment.

  Further, by including the effective timing of at least one or more XY scanners 22 in the effective timing of the Z stage 27, the sample A can be reliably irradiated with the laser beam during the effective timing of the Z stage 27. Thus, scanning by the XY scanner 22 can be performed, and the resolution of the sample A image can be improved.

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.
For example, in the present embodiment, an example in which three laser light sources are provided has been described, but two or four or more laser light sources may be provided. Moreover, it is good also as providing one laser light source with a variable wavelength.

  Further, in the present embodiment, instead of the laser light control units 14, 15, and 16, the laser light sources 11, 12, and 13 may have a function of switching on / off the laser light and switching the wavelength.

A Sample (specimen)
DESCRIPTION OF SYMBOLS 1 Laser scanning microscope 10 Light source unit 11,12,13 Laser light source 14,15,16 Laser light control part 17 Optical coupler 20 Optical unit 21 Spectroscopic part 22 XY scanner (1st scanning part)
23 X galvanometer mirror 24 Y galvanometer mirror 25 Detector 27 Z stage (second scanning unit)
30 Control unit 31 Display device 32 Input unit

Claims (5)

A laser light source for emitting laser light;
At least one resonant galvanometer mirror, a first scanning unit for scanning the laser beam on the specimen from the laser light source for generating the observation image,
A second scanning unit that changes the focal position of the laser beam with respect to the specimen in the optical axis direction so as to change the observation depth of the specimen;
A control unit that controls the emission timing of the laser light source and the scanning timing of the first scanning unit and the second scanning unit;
The control unit changes the effective timing determined by the synchronization signal of the first scanning unit that is turned on within the effective scanning range for each scanning of each line of the first scanning unit and the observation that is changed by the second scanning unit The laser beam is emitted from the laser light source at both effective timings determined by the synchronization signal of the second scanning unit which is turned on every time the depth reaches the focal position where laser scanning by the first scanning unit is to be performed. A laser scanning microscope that performs two-dimensional scanning in a plane along the optical axis of the laser light by repeating the emission control . The laser scanning microscope according to claim 1, wherein the laser light source switches a wavelength of emitted laser light. During said effective timing of the second scanning unit, at least one of the first laser scanning microscope according to claim 1 or claim 2 contains valid timing of the scanning unit. The effective timing of the second scanning unit, said time is determined by the time from the start of the movement of a focal position in the optical axis direction of laser light, the laser scanning microscope according to claim 1. The laser scanning microscope according to claim 1 , wherein the effective timing of the second scanning unit is determined by a pulse signal or a feedback signal corresponding to an observation depth.

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