JP5133600B2 - Laser scanning microscope and microscope observation method - Google Patents

Description

  The present invention relates to a laser scanning microscope, and more particularly to a laser scanning microscope and a microscope observation method for scanning stimulation laser light using an acousto-optic effect.

Conventionally, a laser scanning microscope including an observation scanning optical system for observing a specimen and a stimulation scanning optical system for applying a light stimulus to the specimen is known (for example, see Japanese Patent Laid-Open No. 10-206742). ).
In such a laser scanning microscope, the stimulation laser light emitted from the stimulation laser light source is scanned two-dimensionally by a scanner using a galvanometer mirror, and the sample is irradiated with light to stimulate the light stimulation region. Giving.
Japanese Patent Laid-Open No. 10-206742

  By the way, it is preferable to apply the above-described light stimulation substantially simultaneously to the entire region of the light stimulation region. However, in the above-described conventional laser scanning microscope, since the stimulation laser beam is scanned two-dimensionally on the specimen by the galvanometer mirror and the light stimulation is performed, it takes time from the start to the end of the light stimulation. was there.

  The present invention has been made in view of the above-described circumstances, and provides a laser scanning microscope and a microscope observation method capable of shortening the light stimulus execution time from the start to the end of the light stimulus in the light stimulus region. It is an object.

In order to solve the above problems, the present invention employs the following means.
The present invention two-dimensionally irradiates a specimen placed on a stage with an observation laser beam for observing the specimen, detects the light emitted from the specimen, constructs and displays a fluorescence image A laser scanning microscope that emits stimulation laser light for applying a light stimulus to the specimen, a stimulation scanning unit that scans the stimulation laser light, and the stimulation scanning unit And a control lens for condensing the stimulation laser beam scanned by the stimulation scanning unit and irradiating the sample with the objective laser beam. Comprising at least one acousto-optic element disposed on an optical path, wherein the control unit determines a plurality of frequencies based on a position and a range of a region to which light stimulation is applied, and the high-frequency signals of the determined plurality of frequencies are Acousto-optic element Respect vibrator being kicked by applying simultaneously, laser scanning for morphism irradiation the stimulus laser beam diffracted simultaneously by a plurality of diffraction angles corresponding to each frequency of the high frequency signal to the same time the region Providing a scanning microscope.

According to the above configuration, the stimulation laser light emitted from the stimulation laser light source is scanned by the acousto-optic element included in the stimulation scanning unit, and is irradiated onto the sample via the objective lens. In this case, high-frequency signals having a plurality of frequencies determined based on the position and range of the photostimulation region are simultaneously applied from the control unit to the vibrator attached to the acoustooptic device.
When high-frequency signals having a plurality of frequencies are simultaneously applied to the vibrator, the stimulation laser light incident on the acousto-optic element is simultaneously diffracted at a diffraction angle corresponding to each frequency of the high-frequency signal. The light is emitted as a plurality of spot lights arranged in the direction. Here, as the frequency interval of the high-frequency signal applied to the vibrator is narrower, the distance between the spot lights arranged in a row is also narrower. As a result, by adjusting the frequency interval of the high-frequency signal, the interval of the spot light can be adjusted, and by narrowing the frequency interval to some extent, a portion overlapping each spot light arranged in a row is provided. It is also possible to generate a line-shaped laser beam.
According to the present invention, it is possible to simultaneously irradiate a specimen with a plurality of spot lights, so that light stimulation can be performed more efficiently than when a single spot light is scanned two-dimensionally. Can do.

  In the laser scanning microscope, the stimulation scanning unit includes a first acoustooptic device that diffracts incident stimulation laser light in a first direction, and the incident stimulation laser light in the first direction. It is good also as comprising the 2nd acousto-optic element made to diffract in the direction which crosses.

  According to the above configuration, the stimulation laser light emitted from the stimulation laser light source is intersected with the first direction and the first direction by the first and second acoustooptic elements, for example, the horizontal direction and the vertical direction. Will be diffracted. Accordingly, for example, when high-frequency signals having a plurality of frequencies are simultaneously applied to the vibrators attached to the first and second acousto-optic elements, the laser light having a two-dimensional distribution. Can be generated. As a result, since a two-dimensional area can be irradiated simultaneously, light stimulation can be performed more efficiently.

  In the laser scanning microscope, a two-dimensional pattern on a specimen can be obtained by simultaneously applying high-frequency signals having a plurality of frequencies to the respective transducers of the first acoustooptic element and the second acoustooptic element. It is good also as irradiating the laser beam for a stimulus to the area | region simultaneously. If it does in this way, it will become possible to irradiate the laser beam for stimulation efficiently to two-dimensional fields, such as a rectangle, for example.

In the laser scanning microscope, a linear region on the specimen can be obtained by simultaneously applying high-frequency signals having a plurality of frequencies to one transducer of the first acoustooptic element and the second acoustooptic element. It is good also as irradiating the laser beam for stimulation simultaneously. If it does in this way, it will become possible to irradiate the laser beam for stimulation efficiently to the line segment of predetermined length, for example.

  In the laser scanning microscope, a high-frequency signal having a plurality of frequencies is simultaneously applied to one transducer of the first acoustooptic device and the second acoustooptic device, and at least one is applied to the other transducer. By applying a high-frequency signal having a frequency that changes with time, an acoustooptic device driven by the other transducer in a linear irradiation region emitted from the acoustooptic device driven by the one transducer The two-dimensional region on the specimen may be irradiated with the stimulation laser beam.

  In the laser scanning microscope, the frequency of the high-frequency signal applied to the one vibrator may change with time. In this way, for example, it becomes possible to efficiently irradiate the stimulation laser beam to a two-dimensional region such as a circle.

  In the laser scanning microscope, the stimulation scanning unit is incident on an acoustooptic element that diffracts incident stimulation laser light in a horizontal direction or a vertical direction, and in a direction substantially orthogonal to the diffraction direction of the acoustooptic element. And a scanning mirror that scans the stimulated laser beam.

  According to such a configuration, a plurality of spot lights in which stimulation laser lights that are spot lights are arranged in one direction by an acousto-optic element (if the frequency interval of the high-frequency signals is narrow to some extent, the line-shaped laser lights In addition, the optical stimulation laser beam can be scanned in a direction orthogonal to the one direction by the scanning mirror. Thereby, it is possible to scan a plurality of spot lights in the direction orthogonal to the arrangement on the specimen. As a result, light stimulation can be performed efficiently. Also, by changing the frequency of the high-frequency signal applied to the vibrator attached to the acousto-optic device according to the shape of the photostimulation region, it can be applied not only to squares but also to various shapes such as circles and ellipses. Light stimulation can be performed efficiently.

  In the laser scanning microscope, the laser beam for stimulation may be simultaneously applied to a linear region on the specimen by simultaneously applying high-frequency signals having a plurality of frequencies to the vibrator of the acoustooptic device. . The straight region is, for example, a line segment having a predetermined length.

  In the laser scanning microscope, a high-frequency signal having a plurality of frequencies is simultaneously applied to the vibrator of the acoustooptic device, and the irradiation position of the linear region irradiated simultaneously by the acoustooptic device is scanned by a scanning mirror. Accordingly, the two-dimensional region on the specimen may be irradiated with the stimulation laser beam.

  In the laser scanning microscope, a frequency of a high frequency signal applied to the vibrator may be changed with time. By doing so, for example, it becomes possible to efficiently irradiate a stimulation laser beam to a two-dimensional region such as a circle.

  In the laser scanning microscope, the control unit may determine the frequency of the high-frequency signal in consideration of the wavelength of the stimulation laser beam.

  According to such a configuration, since the frequency of the high-frequency signal is determined according to the wavelength of the stimulation laser beam, even when the wavelength of the stimulation laser beam is switched, it is incident at a desired diffraction angle. Light can be diffracted and a certain scanning accuracy can be maintained.

  In the laser scanning microscope, the control unit may correct the amplitude of the high-frequency signal according to a diffraction angle characteristic of the diffracted light intensity of the acoustooptic device.

  According to such a configuration, the amplitude of the high-frequency signal is corrected according to the diffraction angle characteristic of the diffracted light intensity of the acoustooptic device, so that the light intensity of the stimulation laser beam emitted from the acoustooptic device is set to a desired value. The strength can be controlled. This makes it possible to make the light intensity of the stimulation laser light uniform over the entire region of the light stimulation region or to locally vary the light intensity of the stimulation laser light.

  The laser scanning microscope may include a scanning unit that scans the observation laser light on the specimen.

  In the laser scanning microscope, the region irradiated with the stimulation laser beam may be designated on an image acquired in advance by irradiation with the observation laser beam.

  In the laser scanning microscope, a region where the stimulating laser beam is simultaneously irradiated may be spatially continuous by narrowing an interval of a plurality of frequencies of the high-frequency signal from a predetermined value.

  In the laser scanning microscope, the control unit applies different high-frequency signals having different amplitudes for each of the determined frequencies to the vibrator, thereby changing the irradiation intensity of the stimulation laser light for each region. It is also possible to make it.

The present invention two-dimensionally irradiates a specimen placed on a stage with an observation laser beam for observing the specimen, detects the light emitted from the specimen, constructs and displays a fluorescence image A method of observing a microscope, wherein at least one acoustooptic element is disposed on an optical path of a stimulation laser beam for applying an optical stimulus to the specimen, and the vibrator is attached to each acoustooptic element. The stimulating laser light diffracted simultaneously at a plurality of diffraction angles according to each frequency of the high-frequency signal by simultaneously applying a high-frequency signal having a plurality of frequencies determined according to the position and range of the photostimulation region Is provided for simultaneously irradiating the photostimulation region .

According to such a method, the stimulation laser light emitted from the stimulation laser light source is scanned by the acousto-optic element included in the stimulation scanning unit, and is irradiated onto the specimen via the objective lens. In this case, high-frequency signals having one or a plurality of frequencies determined based on the position and range of the photostimulation region are simultaneously applied to the vibrator attached to the acoustooptic device.
When high-frequency signals having a plurality of frequencies are simultaneously applied to the vibrator, the stimulation laser light incident on the acousto-optic element is simultaneously diffracted at a diffraction angle corresponding to each frequency of the high-frequency signal. The light is emitted as a plurality of spot lights arranged in the direction. Here, as the frequency interval of the high-frequency signal applied to the vibrator is narrower, the distance between the spot lights arranged in a row is also narrower. As a result, by adjusting the frequency interval of the high-frequency signal, the interval of the spot light can be adjusted, and by narrowing the frequency interval to some extent, a portion overlapping each spot light arranged in a row is provided. It is also possible to generate a line-shaped laser beam.
According to the present invention, it is possible to simultaneously irradiate a specimen with a plurality of spot lights, so that light stimulation can be performed more efficiently than when a single spot light is scanned two-dimensionally. Can do.

The present invention includes an observation scanning optical system that two-dimensionally irradiates a specimen placed on a stage with an observation laser beam for observing the specimen, and a light detection unit that detects light emitted from the specimen. And a stimulus scanning optical system for applying a light stimulus to the specimen, and the stimulus scanning optical system includes at least one acousto-optic element. A process for setting a diffraction range of the acoustooptic device according to a position and a range of a light stimulation area, and a process for obtaining one or a plurality of frequencies corresponding to the diffraction range double if, at the same time a high-frequency signal having the one or more frequencies, by applied to the vibrator attached to the acoustooptic device, corresponding to each frequency of the high frequency signal Diffraction angle that provides the light stimulus control program for executing diffracted the stimulus laser beam simultaneously and processing for simultaneously irradiated on the computer to said optical stimulation region of.

  By executing such a photostimulation control program by hardware resources, the diffraction range of the acoustooptic device is set according to the position and range of the photostimulation region, and one or a plurality of frequencies corresponding to the diffraction range are set. The high-frequency signal having one or a plurality of frequencies is calculated and applied to the vibrator attached to the acoustooptic device at the same time. As a result, the stimulating laser light incident on the acoustooptic device is simultaneously diffracted at a diffraction angle corresponding to each frequency of the high-frequency signal. As a result, a plurality of spot laser beams arranged in one direction, or a high-speed scanning using a line-shaped stimulation laser beam or an acousto-optic effect depending on the frequency interval of the high-frequency signal. It is possible to irradiate the specimen with the stimulation laser beam. As a result, light stimulation can be performed efficiently.

  According to the present invention, there is an effect that the light stimulus execution time from the start to the end of the light stimulus in the light stimulus region can be shortened.

  Hereinafter, embodiments of a laser scanning microscope according to the present invention will be described with reference to the drawings.

[First Embodiment]
As shown in FIG. 1, the laser scanning microscope according to the present embodiment is a laser scanning confocal microscope. In FIG. 1, optical components such as various lenses and pinholes are omitted for simplification of description.
As shown in FIG. 1, the laser scanning microscope 1 according to the first embodiment of the present invention scans the observation scanning optical system 2 that scans the observation laser light L1 and the stimulation laser light L2. The scanning optical system 3 for stimulation, the dichroic mirror 4 that combines the observation laser light L1 and the stimulation laser light L2, and the combined observation laser light L1 and stimulation laser light L2 are collected to collect the specimen P. On the other hand, by irradiating the specimen P with the observation laser light L1, the objective lens 5 for condensing the fluorescence F generated by exciting the fluorescent substance in the specimen P is collected by the objective lens 5. And a photodetector 6 for detecting the fluorescence F.

  The observation scanning optical system 2 two-dimensionally scans the observation laser light source 8 that emits the observation laser light L1 and the observation laser light L1 emitted from the observation laser light source 8 in a direction that intersects the optical axis. The first scanner 9 is provided.

Between the observation laser light source 8 of the observation scanning optical system 2 and the first scanner 9, the light is generated in the specimen P, collected by the objective lens 5, and passes through the dichroic mirror 4 and the first scanner 9. A dichroic mirror 12 for branching the returning fluorescent light F from the observation laser beam L1 to the photodetector 6 is provided.
The first scanner 9 includes a first galvanometer mirror 9a that scans the observation laser light L1 in the horizontal direction corresponding to the left-right direction of the fluorescent image, which will be described later, and the observation laser in the vertical direction corresponding to the vertical direction of the fluorescence image. This is a so-called proximity galvanometer mirror including a second galvanometer mirror 9b that scans the light L1. Thereby, the observation laser beam L1 is two-dimensionally scanned on the specimen P by the raster scan method.

  The stimulation scanning optical system 3 is two-dimensionally arranged so that the stimulation laser light source 15 that emits the stimulation laser light L2 and the stimulation laser light L2 emitted from the stimulation laser light source 15 intersect the optical axis. And a second scanner (stimulation scanning unit) 17 for scanning. Details of the stimulation scanning optical system 3 will be described later.

The photodetector 6 includes a confocal pinhole 31, dichroic mirrors 32 and 33, a first light detection unit 34, and a second light detection unit 35.
The first light detection unit 34 includes a dispersion element 36, a lens 37, a slit 38, and a photoelectric conversion element 39. The second light detection unit 35 includes a photometric filter 40 and a photoelectric conversion element 41. In the present embodiment, a planar diffraction grating is used as the dispersive element 36, and the center of the wavelength band to be detected is selected by rotating the planar diffraction grating. The photometric filter 40 has a characteristic of transmitting only light of a specific wavelength.

Each photoelectric conversion element 39, 41 is connected to the control device 20 via an A / D converter (not shown). The control device 20 includes a storage device 21, a CPU 22, and a memory 23. The control device 20 may be constituted by a so-called personal computer. The CPU 22 implements various control contents by reading various data and various programs stored in the storage device 21 into the memory 23 and executing information processing / calculation processing. The storage device 21 is, for example, an HD (Hard Disc), a ROM (Read Only Memory), or the like. The memory 23 is, for example, a RAM (Random Access Memory).
The CPU 22 generates a two-dimensional fluorescence image based on the scanning position information on the specimen P of the observation laser light L1 by the first scanner 9 and the light intensity information of the fluorescence F detected by the photodetector 6. It is constructed and displayed on the display device 24.
An input device 25 is connected to the control device 20. The input device 25 is used for various input operations by an operator.

In the stimulation scanning optical system 3, the second scanner 17 includes a first acoustooptic element 17a that diffracts the stimulation laser beam L2 in the horizontal direction corresponding to the left-right direction of the fluorescence image, which will be described later, and the upper and lower sides of the fluorescence image. And a second acousto-optic element 17b that diffracts the stimulation laser beam L2 in the vertical direction corresponding to the direction.
In the present embodiment, the first acoustooptic element 17a and the second acoustooptic element 17b are acousto optical deflectors (AOD).
The acousto-optic elements 17a and 17b are provided with vibrators (vibration supply units) 18a and 18b that are driven by a high-frequency signal applied by a scanning control unit 19 described later.
The scanning control unit 19 is connected to the control device 20 and applies high-frequency signals in a frequency band corresponding to signal control information from the control device 20 to the vibrators 18a and 18b.

  The storage device 21 of the control device 20 stores the correspondence between the first-order diffraction angle θ and the frequency f of each acousto-optic element 17a, 17b. For example, the scanning control unit 19 stores a relational expression represented by the following expression (1).

  θ = λ · f / Va (1)

In the above equation (1), θ is an angle (hereinafter referred to as “diffraction angle”) formed by the reference lines of the acoustooptic elements 17a and 17b and the first-order diffracted light, as shown in FIG. Is the wavelength of the laser beam L2, f is the frequency of the high-frequency signal given to the vibrator, and Va is the speed of sound in the crystal in the acoustooptic elements 17a and 17b. This Va has a solid difference and is a value that varies depending on the individual acoustooptic elements 17a and 17b.
Further, the control device 20 stores each pixel position of the fluorescent image displayed on the display device 24 and the diffraction angle θ in each acoustooptic element 17a, 17b in association with each other. Thereby, when the light stimulation area is designated by the operator on the fluorescent image displayed on the display device 24, each acousto-optic is so arranged that the CPU 22 irradiates the laser light L2 for stimulation to the light stimulation area. It is possible to determine the range of the diffraction angle θ of the elements 17a and 17b, and simultaneously apply high-frequency signals having a plurality of frequencies corresponding to the range of the diffraction angle θ to the transducers 18a and 18b via the scanning control unit 19. Become.

As a result, a change in refractive index corresponding to the vibration of each transducer 18a, 18b is generated in the first and second acoustooptic elements 17a, 17b. As shown in FIG. By simultaneously diffracting the incident stimulation laser light L2 at different diffraction angles in the horizontal direction, a plurality of spot lights arranged in the horizontal direction, in other words, spot lights having a one-dimensional distribution can be obtained. Note that the shorter the frequency interval of the high-frequency signal applied to the transducer 18a, the shorter the distance between the spot lights. Therefore, by narrowing the frequency interval of the high-frequency signal to be smaller than a certain value, it is possible to make the adjacent spot light have an overlapping portion and to form a linear laser beam extending in the horizontal direction. The same applies to the following second acoustooptic device 17b.
Further, a plurality of spot lights arranged in the horizontal direction are simultaneously diffracted at a plurality of diffraction angles with respect to the vertical direction by the second acousto-optic element 17b, whereby spot lights having a two-dimensional distribution can be obtained. it can.

[Photostimulation area; square]
The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described below. Here, a case where “square” is designated as the photostimulation region B described later will be described.

  First, pre-scanning is performed as a stage before the start of the experiment (step SA1 in FIG. 4). In this pre-scan, the CPU 22 causes the observation scanning optical system 2 and the photodetector 6 to operate to display a fluorescence image of the specimen P on the display device 24. Specifically, the CPU 22 operates the observation laser light source 8 and the first scanner 9. As a result, the observation laser light L1 emitted from the observation laser light source 8 is two-dimensionally scanned by the first scanner 9 and irradiated onto the specimen P through the dichroic mirror 4 and the objective lens 5. When the observation laser light L1 is irradiated onto the specimen P, the fluorescent substance existing in the specimen P is excited by the observation laser light L1, and fluorescence F is generated. The fluorescence F generated in the specimen P returns through the same path as the observation laser light L1 via the objective lens 5, the dichroic mirror 4 and the first scanner 9, and is separated from the observation laser light L1 by the dichroic mirror 12. To the photodetector 6.

  In the photodetector 6, the fluorescence F forms an image at the confocal pinhole 31, enters the dichroic mirror 32, and is branched into two depending on the wavelength. The fluorescence F1 reflected by the dichroic mirror 32 is reflected by the dispersive element 36 at different angles for each wavelength. When the dispersive element 36 is rotated in this state, light at each wavelength center corresponding to the rotation angle of the dispersive element 36 is collected by the lens 37 onto the slit 38, and only light having a wavelength that has passed through the slit 38 is converted into the light conversion element 39. Is incident on.

On the other hand, the fluorescence F <b> 2 that has passed through the dichroic mirror 32 is guided to the second light detection unit 35 by the dichroic mirror 33. In the second light detection unit 35, the fluorescence F <b> 2 is selectively transmitted by the photometric filter 40 with light having a specific wavelength and is incident on the photoelectric conversion element 41.
The fluorescences F1 and F2 incident on the photoelectric conversion elements 39 and 40 are output as electric signals corresponding to the luminance, converted into digital signals by an A / D converter (not shown), and sent to the control device 20. It is done.
In the control device 20, a two-dimensional fluorescent image is constructed based on the digital signal by the CPU 22 and displayed on the display device 24. As a result, the fluorescent image C on the observation surface is displayed on the display device 24 as shown in FIG.

Next, in the fluorescent image C displayed on the display device 24, as shown in FIG. 5, when the image acquisition region A and the light stimulation region B are designated by the operator (step SA2 in FIG. 4), the control device 20 The CPU 22 sets the scanning range of the first scanner 9 according to the designated position and range of the image acquisition area A (step SA3 in FIG. 4), and according to the designated position and range of the photostimulation area B. Then, the frequency of the high frequency signal applied to the vibrators 18a and 18b is calculated (step SA4 in FIG. 4).
Hereinafter, the frequency band setting procedure performed in step SA4 of FIG. 4 will be described in detail with reference to FIG.

First, the CPU 22 of the control device 20 stores the wavelength λ of the stimulation laser light, the acoustic velocity Va1 in the crystal in the first acoustooptic device 17a, and the acoustic velocity Va2 in the crystal in the second acoustooptic device 17b. To the memory 23 (step SB1 in FIG. 6).
Subsequently, the CPU 22 determines the diffraction angle range θh1 to θh2 of the first acoustooptic device 17a based on the horizontal position and range in the photostimulation region B (points Ph1 to Ph2 in FIG. 5) (FIG. 5). 6 step SB2), a plurality of frequencies fh1 to fh2 respectively corresponding to the diffraction angle ranges θh1 to θh2 are determined based on the above equation (1) (step SB3 in FIG. 6).

For example, the frequency fh1 corresponding to the diffraction angle θh1 and the frequency fh2 corresponding to the diffraction angle θh2 are obtained as follows.
fh1 = θh1 · Va1 / λ
fh2 = θh2 · Va1 / λ
Similarly, the CPU 22 determines the diffraction angle range θv1 to θv2 of the second acoustooptic element 17b based on the vertical position and range (points Pv1 to Pv2 in FIG. 5) in the photostimulation region B (FIG. 5). 6 SB4), a plurality of frequencies fv1 to fv2 respectively corresponding to the diffraction angle ranges θv1 to θv2 are determined based on the above equation (1) (step SB5 in FIG. 6).
For example, the frequency fv1 corresponding to the diffraction angle θv1 and the frequency fv2 corresponding to the diffraction angle θv2 are obtained as follows.
fv1 = θv1 · Va2 / λ
fv2 = θv2 / Va2 / λ
In this way, when the CPU 22 obtains a plurality of frequencies based on the position and range of the photostimulation region B, it outputs these information to the scanning control unit 19 (step SB6 in FIG. 6).

  When the image acquisition and the light stimulus preparation are completed in this way, the CPU 22 operates the observation laser light source 8 and scans the first scanner 9 in the above-described scanning range, so that the observation laser light L1 is obtained. By irradiating the image acquisition area A of the sample P two-dimensionally and detecting the fluorescence generated thereby, a fluorescence image in the image acquisition area A is constructed and displayed on the display device 24. In parallel with the image acquisition, the scanning control unit 19 determines a plurality of frequencies in the range of the frequencies fh1 to fh2 and fv1 to fv2 acquired from the CPU 22 at preset frequency intervals, and discretely A high-frequency signal having each distributed frequency is simultaneously applied to each transducer 18a, 18b (step SA5 in FIG. 4). The frequency interval may be uniquely determined in advance, or a plurality of frequency intervals are registered in advance as candidates, and the scan control unit 19 selects and sets one of these candidates. It is good. Further, the operator may set the frequency interval from the input device 25.

  As a result, the vibrators 18a and 18b vibrate based on the high-frequency signals of the respective frequencies, so that the vibrations propagate to the first acousto-optic element 17a and the second acousto-optic element 17b, respectively. Periodic refractive index changes occur in the crystal. When the stimulation laser light source 15 is operated by the CPU 22 in this state, the stimulation laser light L2 is emitted from the stimulation laser light source 15 and is incident on the first acousto-optic element 17a provided on the optical path. Become.

  The stimulating laser light L2 incident on the first acoustooptic device 17a is diffracted almost simultaneously at a diffraction angle corresponding to each frequency of the high-frequency signal (discrete frequency values from fh1 to fh2). As shown in FIG. 3, a plurality of spot lights arranged in the horizontal direction of the photostimulation region B, in other words, a stimulation laser beam L2 having a one-dimensional distribution, is guided to the second acoustooptic device 17b. The one-dimensional distribution of stimulating laser light L2 incident on the second acoustooptic device 17b is diffracted almost simultaneously at a diffraction angle corresponding to each frequency of the high-frequency signal (discrete frequency values from fv1 to fv2). The As a result, a plurality of spot lights arranged in the vertical direction of the photostimulation region B are generated. As shown in FIG. 3, a plurality of spot lights arranged in the horizontal direction and the vertical direction are generated from the second acousto-optic element 17b. The spot light, in other words, the stimulating laser light L2 having a two-dimensional distribution is emitted.

  The stimulation laser light L2 having a two-dimensional distribution is combined with the above-described observation laser light L1 in the dichroic mirror 4 and irradiated onto the specimen P through the objective lens 5. As a result, it is possible to perform optical stimulation on the entire area of the stimulation area B almost simultaneously. When the CPU 22 executes such light stimulation for a designated light stimulation time, the CPU 22 ends the light stimulation. Further, while the light stimulation is being performed, the CPU 22 continuously activates the first scanner 9 and the like of the observation scanning optical system 2, whereby fluorescence images before and after the light stimulation are acquired and continuously displayed on the display device 24. Will be displayed.

[Light stimulation area; oval]
Next, in step SA2 of FIG. 4 described above, the procedure for setting the frequency of the high-frequency signal when an ellipse is designated as the photostimulation region B as shown in FIG. 7 will be described. When an elliptical shape is set as the photostimulation region B, it is impossible to irradiate all the regions at the same time as in the case of the square described above. Therefore, in the case of an elliptical shape, the entire elliptical light stimulation region B is irradiated by scanning the stimulation laser light L2 having a one-dimensional distribution whose irradiation range changes with time. In the present embodiment, the first acoustooptic element 17a is used as a scanning optical element, and the second acoustooptic element 17b is a plurality of spot lights arranged in the vertical direction as stimulation laser light L2 that is spot light. And the distance from end to end of the plurality of spot lights is changed according to time. In the following description, a width from a minimum frequency to a maximum frequency among a plurality of frequencies is defined as a “frequency band”.

First, the CPU 22 of the control device 20 stores the wavelength λ of the stimulation laser light L2, the acoustic velocity Va1 in the crystal in the first acoustooptic element 17a, and the acoustic velocity Va2 in the crystal in the second acoustooptic element 17b. 21 to the memory 23.
Subsequently, the CPU 22 determines a time Tw required to stimulate the entire region of the photostimulation region B, in other words, a photostimulation time Tw from the start to the end of the photostimulation for the photostimulation region B, and this photostimulation time. By obtaining θh (tk) as the diffraction angle of the first acousto-optic element at an arbitrary time tk within Tw, and by changing this tk with a minute time Δt from the light stimulation start time t0 to the end time tw, the light stimulation time The diffraction angle θh (tk: k = 0,..., W) of the first acousto-optic element 17a is obtained every minute time Δt within Tw.

Subsequently, the frequency bands fh (t0),..., Fh (tw) of the high-frequency signal corresponding to the diffraction angle ranges θh (t0),..., Θh (tw) obtained every minute time Δt are described above. (1) It determines based on Formula.
For example, the frequency band fh (tk) of the high-frequency signal corresponding to the diffraction angle θh (tk) at time tk is obtained as follows.
fh (tk) = θh (tk) · Va1 / λ
As a result, as shown in FIG. 8, a high-frequency signal whose frequency increases discretely from fh (t0) to fh (tw) in proportion to time is applied.

  Subsequently, the CPU 22 sets the diffraction angle range θh1 (tk) to θh2 (tk) of the second acoustooptic element at an arbitrary time tk within the photostimulation time Tw to the position and range in the vertical direction of the photostimulation region B. By changing this tk from the light stimulation start time t0 to the end time tw with a minute time Δt, the diffraction angle range θv1 of the second acoustooptic element for each minute time Δt within the light stimulation time Tw is obtained. (T0) to θv2 (t0),... Θv1 (tk) to θv2 (tk)..., Θv1 (tw) to θv2 (tw) are obtained.

Subsequently, the diffraction angle range θv1 (t0) to θv2 (t0) obtained for each minute time Δt (here, θv1 (t0) = θv2 (t0)),... Θv1 (tk) to θv2 (tk) , Θv1 (tw) to θv2 (tw) (here, θv1 (tw) = θv2 (tw)), the frequency band fv1 (t0) to fv2 (t0),. (Tw) to fv2 (tw) are determined based on the above equation (1).
For example, the frequency bands fv1 (tk) to fv2 (tk) of the high-frequency signal corresponding to the diffraction angle range θv1 (tk) to θv2 (tk) at time tk are obtained as follows.
fv1 (tk) = θv1 (tk) · Va2 / λ
fv2 (tk) = θv2 (tk) · Va2 / λ
As a result, as shown in FIG. 9, a high frequency signal whose frequency band varies with time is applied.

In this way, when the CPU 22 obtains the frequency bands of the high-frequency signals to be given to the transducers 18 a and 18 b based on the position and range of the photostimulation region B, the CPU 22 outputs these frequency information to the scanning control unit 19.
As a result, at the time of performing the optical stimulation described above, a high-frequency signal based on the frequency information is applied to the transducers 18a and 18b, and the first and second acoustooptic elements 17a and 17b are corresponding to the high-frequency signal. A refractive index change occurs in the acousto-optic crystal. The stimulating laser light L2 emitted from the stimulating laser light source 15 is incident on the first acoustooptic device 17a, and is diffracted by an angle corresponding to the frequency in the horizontal direction in the fluorescent image to generate the second acoustic signal. The light enters the optical element 17b. In the second acoustooptic device 17b, a plurality of spot lights arranged in the vertical direction of the fluorescent image are generated by being diffracted almost simultaneously in the frequency band fv1 (tk) to fv2 (tk). In other words, it is converted into a one-dimensional distribution of stimulation laser light L2.

  The one-dimensional distribution of the stimulating laser beam L2 is combined with the observation laser beam L1 described above at the dichroic mirror 4 and irradiated onto the specimen P via the objective lens 5. Then, when the frequency and frequency band of the high-frequency signal applied to the first and second vibrators 18a and 18b change with time, the length in the vertical direction on the sample P changes with time. The next-order stimulation laser beam is scanned in the horizontal direction. As a result, the stimulation laser beam L2 is applied to the entire elliptical stimulation region B shown in FIG.

[Light stimulation area; straight line]
Next, in step SA2 described above, the procedure for setting the frequency of the high-frequency signal when a straight line along the horizontal direction of the fluorescent image is designated as the photostimulation region B as shown in FIG.

First, the CPU 22 of the control device 20 stores the wavelength λ of the stimulation laser light L2, the acoustic velocity Va1 in the crystal in the first acoustooptic element 17a, and the acoustic velocity Va2 in the crystal in the second acoustooptic element 17b. 21 to the memory 23.
Subsequently, the CPU 22 determines the diffraction angle range θh1 to θh2 of the first acoustooptic device 17a based on the horizontal position and range in the photostimulation region B (points Ph1 to Ph2 in FIG. 5). The frequency bands fh1 to fh2 of the high frequency signal corresponding to the diffraction angle range θh1 to θh2 are determined based on the above equation (1).

For example, the frequency fh1 corresponding to the diffraction angle θh1 and the frequency fh2 corresponding to the diffraction angle θh2 are obtained as follows.
fh1 = θh1 · Va1 / λ
fh2 = θh2 · Va1 / λ
Similarly, the CPU determines the diffraction angle θv of the second acoustooptic element 17b based on the vertical position in the photostimulation region B, and sets the frequency band fv of the high-frequency signal corresponding to the diffraction angle θv to the above ( 1) Determine based on the equation.
For example, the frequency fv corresponding to the diffraction angle θv is obtained as follows.
fv = θv · Va2 / λ

In this way, when the CPU 22 obtains the frequency bands of the high frequency signals to be given to the transducers 18 a and 18 b based on the position and range of the photostimulation region B, the CPU 22 outputs these pieces of information to the scanning control unit 19.
As a result, at the time of performing the above-described optical stimulation, high-frequency signals of each frequency based on the frequency information are applied to the transducers 18a and 18b, and the first and second acoustooptic elements are corresponding to the high-frequency signals. A refractive index change occurs in the acoustooptic crystals 17a and 17b. The stimulating laser light L2 emitted from the stimulating laser light source 15 is incident on the first acousto-optic element 17a, and is simultaneously diffracted by an angle corresponding to the frequency in the horizontal direction in the fluorescent image, and is one-dimensionally distributed. And is incident on the second acoustooptic device 17b. In the second acoustooptic device 17b, the position of the fluorescent image in the vertical direction is adjusted by diffracting in the frequency band fv. The position-adjusted one-dimensional distribution of the stimulating laser light L2 is combined with the observation laser light L1 described above in the dichroic mirror 4 and irradiated onto the specimen P via the objective lens 5.
As a result, the laser light L2 for stimulation is simultaneously irradiated as a plurality of spot lights over the entire linear light stimulation region shown in FIG.

  As described above, according to the laser scanning microscope 1 according to the present embodiment, the first acoustooptic device in which the stimulation scanning optical system 3 diffracts the incident stimulation laser beam L2 in the horizontal direction. 17a and the second acousto-optic element 17b that diffracts the incident stimulation laser beam in the vertical direction, the sample P can be irradiated with the two-dimensional distribution of the stimulation laser beam L2. . As a result, it is possible to execute light stimulation on the entire region of the light stimulation region B substantially simultaneously. As a result, the light stimulus execution time from the start to the end of the light stimulus can be shortened. Further, by narrowing the frequency interval of the high-frequency signal applied to the vibrators 18a and 18b, it becomes possible to perform more precise light irradiation.

  Further, when the photostimulation region B is set to be a circle or an ellipse, one of the first and second acoustooptic elements 17a and 17b is used as a scanning optical element to stimulate a one-dimensional distribution. By scanning with the laser beam L2 for light, it is possible to perform light stimulation over the entire circular or elliptical light stimulation region B. As described above, even when the stimulation laser beam L2 having a one-dimensional distribution is scanned, the stimulation laser beam is scanned using the acousto-optic effect. Therefore, the stimulation laser beam is utilized using a galvanometer mirror or the like. Compared to the case of scanning, the scanning time can be shortened.

  The correspondence between the diffraction angle characteristics of the acoustooptic elements 17a and 17b, that is, the frequency of the high-frequency signal and the diffraction angle θ of the stimulation laser beam L2 emitted from the acoustooptic elements 17a and 17b is determined by the stimulation laser beam. It varies depending on the wavelength of L2. Therefore, the wavelength of the stimulation laser beam L2 and the diffraction angle characteristic of the acoustooptic device are stored in advance in the storage device 21 in the control device 20, and the diffraction angle characteristic corresponding to the wavelength of the stimulation laser beam L2 is stored. The CPU 22 may determine the frequency range of the high-frequency signal by referring to each time. As described above, when the wavelength of the stimulation laser beam L2 is switched by storing the wavelength of the stimulation laser beam L2 and the diffraction angle characteristics of the acoustooptic elements 17a and 17b in the storage device 21 in association with each other. However, the same position can be irradiated with the stimulation laser beam L2.

In the present embodiment, the case where the photostimulation region B is a quadrangle, a circle, and a straight line has been described. However, for example, when the photostimulation region B such as a curve or a set of a plurality of points is designated, the CPU 22 By determining the frequency or frequency band to be applied to the transducers 18a and 18b according to the position and range of the photostimulation region B, the photostimulation can be similarly performed on the photostimulation region B. .
In this embodiment, the case where the observation scanning optical system 2 and the stimulation scanning optical system 3 are operated simultaneously has been described. However, the present invention is not limited to this operation mode. For example, the stimulation scanning optical system 3 is operated. While there is, the observation scanning optical system 2 may be stopped.
Furthermore, although the case where an acoustooptic polarizer is used as the acoustooptic element has been described in the present embodiment, the present invention is not limited thereto, and other acoustooptic elements such as an acoustooptic modulator may be used.
In the above embodiment, the scanning control unit 19 determines each frequency belonging to this frequency range based on the frequency information given from the CPU 22 at a predetermined frequency interval. The CPU 22 may determine discrete frequencies belonging to each frequency range.

In the present embodiment, the second scan is configured by a combination of the two acoustooptic elements 17a and 17b. Instead, the second scan is configured by either one of the acoustooptic elements 17a and 17b. Also good. In such a configuration, the sample P is irradiated with a one-dimensional stimulation laser beam.
Further, the arrangement of the acoustooptic elements 17a and 17b may be reversed.

[Second Embodiment]
Next, a laser scanning microscope according to the second embodiment of the present invention will be described.
The laser scanning microscope according to the present embodiment is different from the first embodiment in that the CPU 22 also controls the amplitude of the high frequency signal applied to the vibrators 18a and 18b.
Hereinafter, regarding the laser scanning microscope of the present embodiment, description of points that are common to the first embodiment will be omitted, and different points will be mainly described.

  In FIG. 1, acoustooptic elements 17a and 17b employed in the second scanner 17 are diffracted, that is, emitted stimulation laser light according to the frequency band of the high-frequency signal applied to the transducers 18a and 18b. It has the characteristic that the intensity of L2 varies. Here, the intensity of the stimulation laser beam L2 emitted from the acoustooptic elements 17a and 17b and the amplitude of the high-frequency signal applied to the transducers 18a and 18b are in a proportional relationship. Therefore, in the present embodiment, by correcting the amplitude of the high-frequency signal applied to the vibrators 18a and 18b, the variation in the intensity of the laser light generated according to the frequency band of the high-frequency signal is eliminated.

In the laser scanning microscope according to the present embodiment, the storage device 23 stores the diffracted light intensity characteristics of the first and second acoustooptic elements in addition to the information described above (see FIG. 11).
When the CPU 22 sets the frequency or frequency band of the high-frequency signal to be applied to each transducer 18a, 18b according to the photostimulation region B by the same procedure as in the first embodiment, the set frequency or frequency band is set. Accordingly, the amplitude of the high frequency signal is corrected.
Specifically, the amplitude of the high frequency signal is corrected using the following equation (2).

Pf ′ = (Pmax / Pcur) · Pf (2)
In the above equation (2), Pf ′ is the amplitude of the high-frequency signal after correction, Pmax is the maximum intensity of the laser light after diffraction, Pcur is the intensity of the laser light at the frequency to be corrected, and Pf is the high-frequency signal before correction. Of the amplitude.

  The CPU 22 corrects the amplitude of each high-frequency signal using the above (2), and outputs the corrected amplitude information and the set frequency information to the scanning control unit 19. As a result, a high-frequency signal having a corrected amplitude is applied to each transducer 18a, 18b, and the light intensity of the stimulation laser light L2 output from the first and second acousto-optic elements 17a, 17b is: It is possible to maintain the same light intensity regardless of the frequency applied to the vibrator, that is, regardless of the diffraction angle θ of the stimulation laser light L2.

  As described above, according to the laser scanning microscope according to the present embodiment, the amplitude is corrected according to the frequency of the high-frequency signal applied to the transducers 18a and 18b. The intensity of the stimulation laser beam L2 output from 17a and 17b can be made the same without depending on the diffraction angle. Thereby, the sample P can be irradiated with two-dimensional stimulation laser light having uniform intensity.

  In the present embodiment, the case where the intensity of the stimulation laser light L2 is made uniform irrespective of the diffraction angle θ in the acoustooptic elements 17a and 17b has been described. However, the diffracted light intensity characteristic shown in FIG. 11 is used. Thus, the light intensity can be intentionally controlled. Specifically, when it is desired to irradiate a part of the light stimulation region B with light stronger than the other part, the amplitude of the high-frequency signal having a frequency related to that part may be set large. As described above, according to the laser scanning microscope according to the present embodiment, it is possible to improve the accuracy of intensity control of the stimulation laser light L2 irradiated on the specimen P.

  For example, in the rectangular photostimulation region B shown in FIG. 5, when it is desired to change the light intensity with respect to the horizontal direction, the first vibrator 18a of the first acoustooptic element 17a corresponding to the horizontal direction. What is necessary is just to change the amplitude of the high-frequency signal of each frequency given to 1 according to a position.

  For example, as shown in FIG. 12, in the light stimulation region B, when the light intensity is decreased stepwise in the order of the left region A1, the central region A2, and the right region A3, as shown in FIG. In addition, the amplitude of the high-frequency signal having the frequency corresponding to the left region is maximized, the amplitude of the high-frequency signal having the frequency corresponding to the central region A2 is set to an intermediate value, and the high-frequency signal having the frequency corresponding to the right region is The amplitude should be the lowest. Here, the case where it is desired to change the light intensity along the horizontal direction has been described. Similarly, the amplitude of the high-frequency signal applied to the second vibrator 18b corresponding to the second acousto-optic element 17b is the frequency. It is possible to change the light intensity two-dimensionally by changing it according to. Thereby, finer strength control can be realized.

  Further, for example, as shown in FIG. 14, when it is not desired to irradiate the central portion of the elliptical light stimulation region B with the stimulation laser light L2, that is, the stimulation laser light L2 is applied to the donut-shaped light stimulation region B. In the case of irradiation, light irradiation can be avoided by setting the amplitude of the frequency band corresponding to the region where the light stimulation is not desired to be performed to zero in the second acoustooptic device 17b. For example, as shown in FIG. 15, at a certain time tk, in the frequency band from fva to fvd, in the frequency band from fvb to fvc, the region corresponding to this frequency fvb to fvc is set to zero. It is possible to avoid irradiation with the stimulation laser L2. And by changing the frequency band which makes an amplitude zero according to time, it becomes possible to perform light stimulation with respect to the donut-shaped light stimulation area | region B as shown in FIG.

  In this way, by controlling the amplitude of the high-frequency signal, the light intensity at each position is adjusted, so that the light stimulation area B of various shapes including the donut-shaped light stimulation area B as shown in FIG. It becomes possible to cope with. Thereby, even if it is an object of non-geometric shapes, such as a living cell, it becomes possible to perform light stimulation according to the shape. For example, in one whole cell such as a nerve, light stimulation can be performed except for only the cell nucleus.

[Third Embodiment]
Next, a laser scanning microscope according to the third embodiment of the present invention will be described.
The laser scanning microscope according to this embodiment is different from the laser scanning microscope 1 according to the first embodiment in that a galvano mirror 51 is used instead of the second acousto-optic element 17b as shown in FIG. And galvano mirror 51 is mechanically vibrated to deflect light.
The galvanometer mirror 51 scans the stimulation laser beam L2 in which the position of the fluorescent image in the horizontal direction is adjusted by the first acousto-optic element 17a in the vertical direction of the fluorescent image.

  For example, in such a laser scanning microscope, the stimulation laser light L2 emitted from the stimulation laser light source 15 is converted into the stimulation laser light L2 having a one-dimensional distribution in the horizontal direction by the first acoustooptic device 17a. Guided to the galvanometer mirror 51. In the galvanometer mirror 51, the one-dimensional distribution of the stimulation laser beam L 2 is scanned in the vertical direction, guided to the dichroic mirror 4 through the mirror 52, and combined with the observation laser beam L 1 through the objective lens 5. The specimen P is irradiated. As a result, the one-dimensional distribution of the stimulation laser light L2 in the horizontal direction is scanned in the vertical direction, so that the light stimulation can be efficiently performed on the desired light stimulation region B.

  In the above embodiment, the arrangement of the galvano mirror 51 and the first acousto-optic element 17a may be exchanged, and the arrangement of the galvano mirror 51 and the mirror 52 may be exchanged. Further, the second scan 17 may be configured by a combination of the second acoustooptic element 17 b and the galvanometer mirror 51.

  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.

1 is an overall configuration diagram showing a laser scanning microscope according to a first embodiment of the present invention. It is explanatory drawing for demonstrating the effect | action of the acoustooptic device shown in FIG. It is explanatory drawing for demonstrating the effect | action of the 2nd scanner shown in FIG. It is the flowchart which showed an example of the process sequence of the laser scanning microscope which concerns on the 1st Embodiment of this invention. It is the figure which showed an example of the display screen when an image acquisition area and a square photostimulation area | region are designated in the fluorescence image. It is the flowchart which showed an example of the process sequence for determining the frequency of a high frequency signal in the scanning condition setting for stimulation. It is the figure which showed an example of the display screen when the image acquisition area | region and an elliptical light stimulus area | region are designated in the fluorescence image. It is the figure which showed the relationship between the frequency of the high frequency signal applied to a 1st vibrator | oscillator when an elliptical light stimulation area | region is designated, and time. It is the figure which showed the relationship between the frequency of the high frequency signal applied to a 2nd vibrator | oscillator when an elliptical photostimulation area | region is designated, and time. It is the figure which showed an example of the display screen when the image acquisition area | region and an elliptical light stimulus area | region are designated in the fluorescence image. It is the figure which showed an example of the diffracted light intensity characteristic. It is the figure which showed an example in the case of changing the light intensity in a photostimulation area | region. FIG. 13 is a diagram showing a relationship between a frequency and an amplitude applied to the first vibrator when the stimulation laser beam is irradiated so as to have the light intensity shown in FIG. 12. It is the figure which showed the donut type photostimulation area | region. FIG. 15 is a diagram showing the relationship between the frequency and amplitude of a high-frequency signal applied to the second vibrator at time tk when the donut-shaped photostimulation region shown in FIG. 14 is designated. It is the figure which showed an example of the structure of the 2nd scanner in the laser scanning microscope which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

P Specimen F Fluorescence L1 Observation laser light L2 Stimulation laser light 1 Laser scanning microscope 2 Observation scanning optical system 3 Stimulation scanning optical system 4 Dichroic mirror 5 Objective lens 6 Photo detector 8 Observation laser light source 9 First Scanner 9a First galvanometer mirror 9b Second galvanometer mirror 15 Stimulation laser light source 17 Second scanner 17a, 17b Acoustooptic element 18a, 18b Transducer 19 Scan control unit 20 Controller 21 Storage device 22 CPU
23 memory 24 display device 25 input device

Claims (23)

Laser scanning type for irradiating a specimen placed on a stage with an observation laser light for observing the specimen two-dimensionally, detecting the light emitted from the specimen, constructing and displaying a fluorescence image A microscope,
A stimulation laser light source that emits stimulation laser light for applying optical stimulation to the specimen;
A stimulation scanning unit that scans the stimulation laser beam;
A control unit for controlling the stimulation scanning unit;
An objective lens that collects the stimulation laser beam scanned by the stimulation scanning unit and irradiates the sample;
The stimulation scanning unit includes at least one acousto-optic element disposed on an optical path of the stimulation laser beam;
The control unit determines a plurality of frequencies based on a position and a range of a region to which light stimulation is applied, and simultaneously applies high-frequency signals of the determined plurality of frequencies to the vibrator attached to the acoustooptic device. by applying to the high frequency signal a plurality of diffraction angles in the laser scanning microscope for morphism simultaneously light of the diffracted the stimulus laser beam simultaneously in said region corresponding to each frequency. The stimulation scanning unit,
A first acousto-optic element that diffracts incident stimulation laser light in a first direction;
The laser scanning microscope according to claim 1, further comprising: a second acousto-optic element that diffracts the incident stimulation laser light in a direction intersecting the first direction.   A stimulating laser is simultaneously applied to a two-dimensional region on the specimen by simultaneously applying high-frequency signals having a plurality of frequencies to the respective vibrators of the first acoustooptic element and the second acoustooptic element. The laser scanning microscope according to claim 2, which irradiates light.   The laser scanning microscope according to claim 3, wherein the two-dimensional region irradiated with the stimulation laser beam is a rectangle.   By simultaneously applying high-frequency signals having a plurality of frequencies to one transducer of the first acoustooptic element and the second acoustooptic element, the stimulating laser beam is simultaneously applied to a linear region on the specimen. The laser scanning microscope according to claim 2 for irradiation.   The laser scanning microscope according to claim 5, wherein the linear region irradiated with the stimulation laser beam is a line segment having a predetermined length.   A frequency that simultaneously applies a high-frequency signal having a plurality of frequencies to one transducer of the first acoustooptic device and the second acoustooptic device, and at least one frequency changes with time to the other transducer. By applying the high-frequency signal, the linear irradiation region emitted from the acousto-optic device driven by the one transducer is switched by the acousto-optic device driven by the other transducer, thereby The laser scanning microscope according to claim 2, wherein the two-dimensional region is irradiated with stimulation laser light.   The laser scanning microscope according to claim 7, wherein a frequency of a high-frequency signal applied to the one vibrator changes with time.   The laser scanning microscope according to claim 8, wherein the two-dimensional region on the specimen irradiated with the stimulation laser beam is circular. The stimulation scanning unit includes:
An acoustooptic device that diffracts the incident stimulating laser beam in the horizontal or vertical direction;
The laser scanning microscope according to claim 1, further comprising: a scanning mirror that scans the incident stimulation laser beam in a direction substantially orthogonal to a diffraction direction of the acoustooptic device.   11. The laser scanning microscope according to claim 10, wherein a high-frequency signal having a plurality of frequencies is simultaneously applied to the transducer of the acoustooptic device, whereby the stimulation laser beam is simultaneously irradiated onto a linear region on the specimen.   The laser scanning microscope according to claim 11, wherein the linear region irradiated with the stimulation laser beam is a line segment having a predetermined length.   A high-frequency signal having a plurality of frequencies is simultaneously applied to the transducer of the acoustooptic device, and the irradiation position of a linear region that is simultaneously illuminated by the acoustooptic device is scanned by a scanning mirror. The laser scanning microscope according to claim 10, wherein the three-dimensional region is irradiated with stimulation laser light.   The laser scanning microscope according to claim 13, wherein a frequency of a high-frequency signal applied to the vibrator changes with time.   The laser scanning microscope according to claim 14, wherein the two-dimensional region on the specimen irradiated with the stimulation laser light is circular.   The laser scanning microscope according to any one of claims 1 to 15, wherein the control unit determines a frequency of the high-frequency signal in consideration of a wavelength of the stimulation laser beam.   The laser scanning microscope according to any one of claims 1 to 16, wherein the control unit corrects the amplitude of the high-frequency signal in accordance with a diffraction angle characteristic of a diffracted light intensity of the acoustooptic device.   The laser scanning microscope according to claim 1, further comprising a scanning unit that scans the observation laser light on the specimen.   The laser scanning microscope according to any one of claims 1 to 18, wherein the region irradiated with the stimulation laser light can be designated on an image acquired in advance by irradiation with an observation laser light. .   The laser scanning microscope according to any one of claims 1 to 19, wherein a region simultaneously irradiated with the stimulation laser beam is spatially continuous by narrowing an interval between a plurality of frequencies of the high-frequency signal from a predetermined value. .   The said control part varies the irradiation intensity | strength of the said laser beam for stimulation for every said area | region by applying simultaneously the high frequency signal from which an amplitude differs for every said determined several frequency to the said vibrator | oscillator. The laser scanning microscope according to any one of 20. Microscope observation method for constructing and displaying a fluorescence image by two-dimensionally irradiating a specimen placed on a stage with a laser beam for observation for observing the specimen, detecting light emitted from the specimen Because
At least one acoustooptic element is arranged on the optical path of the stimulation laser beam for applying the light stimulus to the specimen, and the position and range of the photostimulation region with respect to the vibrator attached to each acoustooptic element By simultaneously applying a high-frequency signal having a plurality of frequencies determined according to the frequency, the stimulation laser beam simultaneously diffracted at a plurality of diffraction angles corresponding to each frequency of the high-frequency signal is simultaneously irradiated onto the light stimulation region. microscopy methods of. An observation scanning optical system that two-dimensionally irradiates a specimen placed on a stage with an observation laser beam for observing the specimen, a light detection unit that detects light emitted from the specimen, and the specimen A laser scanning microscope having a scanning optical system for stimulating the light and having at least one acousto-optic element for controlling the scanning optical system for stimulation. A light stimulus control program,
A process for setting a diffraction range of the acoustooptic device according to the position and range of the photostimulation region,
Processing for obtaining one or more frequencies corresponding to the diffraction range;
By simultaneously applying a high-frequency signal having one or a plurality of frequencies to a vibrator attached to the acoustooptic device, the signals are simultaneously diffracted at a plurality of diffraction angles corresponding to each frequency of the high-frequency signal. A photostimulation control program for causing a computer to execute a process of simultaneously irradiating the photostimulation laser beam with the stimulation laser beam .

Images