JP6177007B2 - Microscope system - Google Patents

Description

  The present invention relates to a microscope system.

  Conventionally, a scanning microscope that irradiates a sample with laser light and observes the reaction of the sample is known (for example, see Patent Document 1). The scanning microscope described in Patent Document 1 includes a shutter in which the stimulation optical system and the observation optical system can switch the passage / blocking of laser light by controlling the diffraction angle, like an acousto-optic element. By switching the passage / blocking of the laser light by the above, the specimen is optically stimulated at an arbitrary timing by the stimulation optical system while acquiring the specimen image by the observation optical system.

JP 2003-315681 A

  However, since the acousto-optic element generates a weak leakage light component of the laser beam regardless of the control of the diffraction angle, the laser beam cannot be completely switched between passing and blocking. Therefore, in the scanning microscope described in Patent Document 1, the reagent reacts at a timing other than the desired timing for optical stimulation of the specimen due to the leaked light component of the laser light at the acoustooptic device, and the light is emitted at the desired timing. There is an inconvenience that it cannot be stimulated.

  On the other hand, as a shutter, there is a mechanical shutter capable of switching between passing / blocking of laser light by moving between a closed position for closing the optical path of the laser light and an open position for opening. However, although the mechanical shutter can reliably block the laser beam by closing the optical path, there is a time delay between the output of the opening / closing operation command and the actual closing of the optical path. There is an inconvenience that passing / blocking cannot be switched instantaneously.

  The present invention has been made in view of the above-described circumstances, suppresses the sample from being irradiated with light at a timing other than a desired timing, and turns on / off the irradiation of light to the sample at an arbitrary timing. It aims at providing the microscope system which can be switched by.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source for generating light, an irradiation optical system for irradiating the light emitted from the light source to the specimen, with respect to light emitted from the light source, you angle incident on the irradiation optical system and acousto-optic device which can switch on and the OFF state you angle does not enter the oN state, removably disposed in the optical path between the illumination optical system and the light source, closing the open state to open the optical path A mechanical shutter that can be switched to a closed state, and a control unit that controls ON / OFF of light irradiation to the specimen by the acoustooptic device and the mechanical shutter, the control unit comprising the acoustooptic device A microscope system that switches the mechanical shutter to an open state before switching to an ON state.

  According to the present invention, the light emitted from the light source is irradiated onto the specimen by the irradiation optical system via the acousto-optic element and the mechanical shutter. In the acousto-optic device, switching between the ON state and the OFF state to change the diffraction angle of light can switch the passage / blocking of light incident on the illumination optical system. In the mechanical shutter, passing / blocking of light incident on the illumination optical system is switched by switching between an open state and a closed state to open / close the optical path. Therefore, ON / OFF of the light irradiation to the specimen can be arbitrarily switched by the control unit.

  In this case, the acoustooptic device can instantaneously switch between the ON state and the OFF state, but a weak light leakage component of light is generated regardless of the control of the diffraction angle. The shut-off cannot be switched completely. On the other hand, the mechanical shutter can completely switch between passing and blocking light, but there is a time delay from the output of the insertion / removal operation command to the actual opening / closing of the optical path.

  Therefore, the control unit switches the mechanical shutter to the open state before switching the acousto-optic element to the ON state, so that the light is almost certainly blocked by the mechanical shutter except for the timing of irradiating the sample with light, and the light is applied to the sample. At the irradiation timing, the light can be transmitted at a desired timing by the acoustooptic device.

  Therefore, the generation of leakage light by the sound optical element and the time delay of the mechanical shutter are compensated for each other to suppress the sample from being irradiated with light at a timing other than the desired timing, and the light irradiation to the sample is turned on. / OFF can be switched at an arbitrary timing.

  In the above-described invention, the control unit inputs the signal of the insertion / removal operation to the mechanical shutter and the time delay required until the mechanical shutter is detached from the optical path before the acoustooptic device. It is also possible to start switching the mechanical shutter to the open state.

  With this configuration, the time difference between the switching from light blocking to passing by the acousto-optic element and the switching from light blocking to passing by the mechanical shutter is minimized, and leakage of the acousto-optic element irradiated to the specimen is achieved. The light component can be reduced.

In the above invention, the control unit, the acousto-optic element at the same time as the switch to OFF state, the mechanical shutter may be begin switching to the closed state.
With this configuration, when the light irradiation to the specimen is stopped, the light is blocked at a desired timing by the acousto-optic device, and after the light irradiation to the specimen is stopped, the light is almost surely received by the mechanical shutter. Can be blocked.

In the above invention, the control unit, the than 2 times the delay time of the mechanical shutter longer OFF state of the acousto-optic device, open the ON state / OFF state and the mechanical shutter of the acousto-optic device The state / closed state may be switched repeatedly.
With this configuration, it is possible to intermittently irradiate the sample with light while suppressing the sample from being irradiated with light at a timing other than the desired timing.

  The above invention comprises a plurality of the light sources that generate light of different wavelengths, and an optical path synthesis unit that synthesizes an optical path with the light emitted from the plurality of light sources, and each of the light sources and the optical path synthesis unit A plurality of the mechanical shutters disposed therebetween may be provided, and the acoustooptic device may be disposed on an optical path of the light combined by the optical path combining unit.

  With this configuration, it is possible to reliably switch light passage / blocking for each light source by each mechanical shutter. The light is almost certainly blocked by the mechanical shutter except for the timing when the sample is irradiated with light from one of the light sources, while the light is transmitted at a desired timing by the acousto-optic device when the light is irradiated from the light source to the sample. Can be made.

  The present invention adjusts the intensity of a laser beam in accordance with a driving voltage and can switch between an ON state in which the laser beam is generated and an OFF state in which the laser beam is not generated, and a laser beam emitted from the laser source. An irradiation optical system that irradiates, a mechanical shutter that is detachably provided in an optical path between the laser light source and the illumination optical system, and that can be switched between an open state that opens the optical path and a closed state that closes the optical path; A control unit that controls ON / OFF of laser light irradiation to the specimen by a laser light source and the mechanical shutter, and the control unit opens the mechanical shutter before switching the laser light source to the ON state. A microscope system for switching is provided.

  According to the present invention, the sample is irradiated with the laser light emitted from the laser light source by the irradiation optical system via the mechanical shutter. In the laser light source, the generation timing of the laser light is switched by adjusting the intensity of the laser light by switching between the ON state and the OFF state. In the mechanical shutter, passing / blocking of the laser light incident on the illumination optical system is switched by switching between an open state and a closed state to open / close the optical path. Therefore, ON / OFF of the laser beam irradiation to the specimen can be arbitrarily switched by the control unit.

  In this case, the laser light source can be switched instantaneously between the ON state and the OFF state, but even in the OFF state, a weak leakage light component of the laser light is generated, so the generation timing of the laser light is completely set. I can't switch.

  Therefore, the control unit switches the mechanical shutter to the open state before switching the laser light source to the ON state, so that the laser light is almost surely cut off by the mechanical shutter except for the timing of irradiating the sample with the laser light. Laser light can be generated at a desired timing from the laser light source at the timing of light irradiation.

  Therefore, the generation of leakage light from the laser light source and the time delay of the mechanical shutter are compensated for each other, and the sample is prevented from being irradiated with laser light at a timing other than the desired timing, and the sample is irradiated with laser light. Can be switched at any timing.

  In the above-described invention, the control unit inputs the mechanical shutter before the laser light source by a time delay required for the mechanical shutter to be detached from the optical path after inputting the insertion / removal operation signal to the mechanical shutter. It is also possible to start switching the shutter to the open state.

  With this configuration, the timing difference between the timing of generating the laser light from the laser light source and the switching of the passage from the interruption of the laser light by the mechanical shutter is minimized, and the leakage light component of the laser light source irradiated to the specimen is reduced. Can be less.

In the above invention, the control section, at the same time as the switch between the laser light source to the OFF state, the mechanical shutter may be begin switching to the closed state.
With this configuration, the generation of laser light is stopped at a desired timing by the laser light source at the timing of stopping the irradiation of the laser light on the specimen, and the mechanical shutter is used after the irradiation of the laser light on the specimen is stopped. Laser light can be blocked almost certainly.

In the above invention, the control unit may make the OFF state of the laser light source longer than the time twice the delay time of the mechanical shutter , and repeatedly switch the ON / OFF state of the laser light source.
With this configuration, it is possible to intermittently irradiate the sample with light while suppressing the sample from being irradiated with light at a timing other than the desired timing.

  In the above-mentioned invention, the image acquisition unit that images the return light from the sample and acquires the image of the sample is provided, and the control unit performs the imaging of the return light before the image acquisition unit starts the imaging. The mechanical shutter may be switched to the open state.

  With this configuration, light can be blocked almost certainly by the mechanical shutter until the timing when the image is acquired by the image acquisition unit. As a result, it is possible to minimize the waste of irradiating the sample with light while the image is not acquired.

  According to the present invention, it is possible to suppress the sample from being irradiated with light at a timing other than a desired timing, and to turn ON / OFF the irradiation of light to the sample at an arbitrary timing.

1 is a schematic configuration diagram showing a microscope system according to a first embodiment of the present invention. It is a schematic block diagram of the acoustooptic device of FIG. (A) is a figure which shows the state which closed the optical path with the mechanical shutter of FIG. 1, (b) is a figure which shows the state which open | released the optical path with the mechanical shutter. It is a block diagram which shows the structure of the control apparatus of FIG. It is a figure explaining the case where a raster scan is performed by the observation scanning unit of FIG. 1, and a tornado scan is performed by the stimulus scanning unit. It is a figure explaining the switching timing of an acousto-optic device and a mechanical shutter. It is a figure explaining the case where a point scan is carried out by a stimulus scanning unit. It is a figure explaining the switching timing of an acousto-optic device and a mechanical shutter. It is a figure explaining the case where a raster scan is carried out by a stimulus scanning unit. It is a figure explaining the switching timing of an acousto-optic device and a mechanical shutter. It is a figure explaining the switching timing of the acousto-optic device and mechanical shutter of the microscope system which concerns on the 1st modification of 1st Embodiment of this invention. It is a figure explaining the structure with which the microscope system which concerns on the 2nd modification of 1st Embodiment of this invention is equipped with three light sources. It is a schematic block diagram which shows the microscope system which concerns on 2nd Embodiment of this invention.

[First Embodiment]
A microscope system according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the microscope system 100 according to the present embodiment generates fluorescence by irradiating the specimen S with excitation light, a stage 1 on which the specimen S is placed, an excitation light source 2 that generates excitation light, and the specimen S. An observation optical system (irradiation optical system) 3, a detection optical system 4 that detects fluorescence generated in the specimen S, a stimulation light source 5 that generates stimulation light, and a stimulus that irradiates the specimen S with stimulation light and provides optical stimulation. An optical system (irradiation optical system) 6 and a control device 7 for controlling these observation optical system 3, detection optical system 4, stimulation optical system 6 and the like are provided.

  Further, the microscope system 100 is incident on the first acousto-optic element 11 and the first mechanical shutter 13 for switching the passage / blocking of the excitation light incident on the observation optical system 3 and the stimulation optical system 6 under the control of the control device 7. A second acousto-optic element 41 and a second mechanical shutter 43 that switch passage / blocking of the stimulation light to be provided are provided.

  The excitation light source 2 generates laser light as excitation light for exciting the fluorescent reagent in the sample S.

  The observation optical system 3 includes an observation scanning unit 15 that deflects excitation light that has passed through the first acoustooptic element 11 and the first mechanical shutter 13, a relay lens 17 that relays excitation light deflected by the observation scanning unit 15, A reflection mirror 19 that reflects the excitation light that has passed through the relay lens 17, an imaging lens 21 that collimates the excitation light reflected by the reflection mirror 19, and the sample S that is irradiated with the excitation light that has passed through the imaging lens 21 On the other hand, an objective lens 23 that collects return light including fluorescence generated in the specimen S is provided.

  The observation scanning unit 15 includes an X-direction scanner 15A and a Y-direction scanner 15B which are provided so as to be swingable about swing axes orthogonal to each other and driven by a raster scan method. These scanners 15 </ b> A and 15 </ b> B are configured to deflect excitation light in two directions orthogonal to each other and scan the sample S two-dimensionally.

  The detection optical system 4 includes a dichroic mirror 31 that branches the fluorescence collected by the objective lens 23 from the optical path of the excitation light, and a photometric filter 33 that selectively passes only the fluorescence wavelength from the return light branched by the dichroic mirror 31. A condensing lens 35 that condenses the fluorescence that has passed through the photometric filter 33, a confocal pinhole 37 that restricts the passage of the fluorescence collected by the condensing lens 35, and the fluorescence that has passed through the confocal pinhole 37. And a photoelectric conversion element 39 for detecting.

  The dichroic mirror 31 is disposed on the optical path between the excitation light source 2 and the observation scanning unit 15, and transmits excitation light from the excitation light source 2, while transmitting excitation light from the sample S via the observation optical system 3. The return light returning from the optical path is reflected toward the photometric filter 33.

The confocal pinhole 37 cuts fluorescence generated at a position shifted in the optical axis direction from the focal position of the excitation light in the sample S out of the fluorescence that has passed through the photometric filter 33, and from the focal position of the excitation light in the sample S. Only the generated fluorescence can pass through.
When detecting the fluorescence, the photoelectric conversion element 39 converts it into a luminance signal corresponding to the luminance and sends it to the control device 7.

  The stimulation light source 5 generates laser light as stimulation light that stimulates the specimen S.

  The stimulation optical system 6 includes a stimulation scanning unit 45 that deflects stimulation light that has passed through the second acoustooptic element 41 and the second mechanical shutter 43, a relay lens 47 that relays stimulation light deflected by the stimulation scanning unit 45, and A dichroic mirror 49 that synthesizes the optical path of the stimulation light transmitted through the relay lens 17 and the optical path of the excitation light of the observation optical system 3 is provided.

  The stimulus scanning unit 45 includes an X-direction scanner 45A and a Y-direction scanner 45B that are driven by a raster scan method around swing axes that are orthogonal to each other. The stimulation scanning unit 45 can deflect the stimulation light incident from the stimulation light source 5 in two directions orthogonal to each other and scan the sample S two-dimensionally.

  The dichroic mirror 49 is disposed on the optical path between the reflection mirror 19 and the imaging lens 21, and reflects the stimulation light transmitted through the relay lens 47 of the stimulation optical system 6 toward the imaging lens 21, Excitation light from the reflection mirror 19 and return light from the imaging lens 21 are transmitted.

The first acoustooptic element 11 and the first mechanical shutter 13 are provided on the optical path between the excitation light source 2 and the observation optical system 3, and the first acoustooptic element 11 and the first mechanical shutter 13 are arranged in this order from the excitation light source 2 side. Has been placed.
The second acoustooptic element 41 and the second mechanical shutter 43 are provided on the optical path between the stimulation light source 5 and the stimulation optical system 6, and the second acoustooptic element 41 and the second mechanical shutter 43 are arranged from the stimulation light source 5 side. Arranged in order.

  As the first acoustooptic device 11, for example, an AOM (Acousto-Optic Modulator) or an AOTF (Acousto-Optics Tunable Filter) can be used. As shown in FIG. 2, the first acoustooptic device 11 induces a change in the refractive index by applying an ultrasonic wave (control signal) to the optical crystal to vibrate the optical crystal with a dense wave. Diffraction of the light beam is caused by using the density as a diffraction grating. In addition, the first acoustooptic device 11 can change the diffraction angle of the light beam by changing the frequency of the control signal to be applied.

The first acousto-optic device 11, the control signals, the ON state of the diffraction angle of the incident excitation light entering from the observation light source 1 to the observation optical system 3, to switch to the OFF state you angle does not enter It can be done.

The second acoustooptic element 41 is configured in the same manner as the first acoustooptic element 11.
The first acoustooptic element 11 and the second acoustooptic element 41 can be instantaneously switched between an ON state and an OFF state by a control signal.

As shown in FIGS. 3A and 3B, the first mechanical shutter 13 is formed by a light shielding member that is detachably provided on the optical path between the first acoustooptic device 11 and the observation optical system 3. Has been. The first mechanical shutter 13 is inserted into the optical path as shown in FIG. 3A by the drive signal to close the optical path, and is removed from the optical path as shown in FIG. 3B to open the optical path. It can be switched to the open state.

The second mechanical shutter 43 is configured in the same manner as the first mechanical shutter 13.
The first mechanical shutter 13 and the second mechanical shutter 43 are from the control device 7 outputting a drive signal until it is actually inserted into or removed from the optical path, that is, from the closed position to the open position or from the open position. There is a time delay before moving to the closed position. Hereinafter, the delay time from when the control device 7 outputs the drive signal to when the first mechanical shutter 13 or the second mechanical shutter 43 actually opens / closes the optical path is referred to as “delay time”.

  As shown in FIG. 4, the control device 7 includes an observation scanning waveform generation circuit 51 and a stimulation scanning waveform generation circuit 52 that send control signals to the observation scanning unit 15 and the stimulation scanning unit 45, and the first acoustooptic device 11 and the second. A first control circuit 53 and a second control circuit 54 for sending a control signal to the acoustooptic device 41; a first drive circuit 55 and a second drive circuit 56 for sending a drive signal to the first mechanical shutter 13 and the second mechanical shutter 43; An image generation unit 57 that generates a two-dimensional image of the specimen S, and a CPU (Central Processing Unit, control unit) 58 that controls these circuits and the image generation unit 57 are provided.

The observation scanning waveform generation circuit 51 generates a control signal for controlling the swing operation of each scanner 15A, 15B to the observation scanning unit 15. The observation scanning waveform generation circuit 51 sends a timing signal for scanning excitation light by the observation scanning unit 15 to the image generation unit 57 via the CPU 58.
The stimulation scanning waveform generation circuit 52 generates a control signal for controlling the swinging operation of each scanner 45A, 45B to the stimulation scanning unit 45.

The first control circuit 53 outputs a control signal for switching between the ON state and the OFF state to the first acoustooptic device 11.
The second control circuit 54 outputs a control signal for switching between the ON state and the OFF state to the second acoustooptic element 41.

The first drive circuit 55 outputs a drive signal for switching between the open state and the closed state to the first mechanical shutter 13.
The second drive circuit 56 outputs a drive signal for switching the open state and the closed state to the second mechanical shutter 43.

  Based on the timing signal of the observation scanning unit 15 sent from the observation scanning waveform generation circuit 51, the image generation unit 57 rearranges the luminance signals sent from the photoelectric conversion element 39 two-dimensionally, and samples S The two-dimensional image is generated. The image of the specimen S generated by the image generation unit 57 is displayed on a monitor (not shown).

  The CPU 58 generates a control signal from the observation scanning waveform generation circuit 51 to control the scanning of the excitation light by the observation scanning unit 15, and also generates a control signal from the stimulation scanning waveform generation circuit 52 to generate the control signal from the stimulation scanning unit 45. The scanning of the stimulus light is controlled.

  Further, the CPU 58 generates a control signal from the control circuit 53 and also generates a drive signal from the drive circuit 55, and turns on the irradiation of the excitation light to the sample S by the first acoustooptic element 11 and the first mechanical shutter 13. / OFF is controlled. Further, the CPU 58 generates a control signal from the control circuit 54 and also generates a drive signal from the drive circuit 56, and turns on the irradiation of the stimulation light to the sample S by the second acoustooptic element 41 and the second mechanical shutter 43. / OFF is controlled.

  The CPU 58 starts to switch the first mechanical shutter 13 from the closed state to the open state first by the delay time before switching the first acoustooptic element 11 from the OFF state to the ON state. Further, the CPU 58 starts switching the first mechanical shutter 13 from the open state to the closed state almost simultaneously with switching the first acoustooptic device 11 from the ON state to the OFF state.

Similarly, before switching the second acoustooptic element 41 from the OFF state to the ON state, the CPU 58 starts to switch the second mechanical shutter 43 from the closed state to the open state first by the delay time. At the same time as switching from the ON state to the OFF state, the second mechanical shutter 43 starts to be switched from the open state to the closed state.
Further, the CPU 58 sends a frame signal for generating an image to the image generation unit 57.

The operation of the microscope system 100 configured as described above will be described.
In order to observe the specimen S with the microscope system 100 according to the present embodiment, first, the specimen S into which the fluorescent indicator is introduced is placed on the stage 1 and excitation light is generated from the excitation light source 2.

  The excitation light emitted from the excitation light source 2 passes through the first acoustooptic element 11 along the optical path incident on the observation optical system 3 when the first acoustooptic element 11 is in the ON state under the control of the CPU 58. When the one acoustooptic element 11 is in the OFF state, it passes through the first acoustooptic element 11 along an optical path that does not enter the observation optical system 3.

  When the first mechanical shutter 13 is in the open state under the control of the CPU 58, the excitation light that has passed through the first acoustooptic element 11 in the ON state passes through the optical path by being opened and enters the observation optical system 3. However, when the first mechanical shutter 13 is in the closed state, the first optical shutter 13 is blocked by closing the optical path.

  Therefore, the CPU 58 can arbitrarily switch ON / OFF the irradiation of the excitation light to the specimen S in the first acoustooptic device 11 and the first mechanical shutter 13.

  The excitation light that has passed through the first acoustooptic device 11 and the first mechanical shutter 13 passes through the dichroic mirror 31 and is deflected by the observation scanning unit 15, then relayed by the relay lens 17 and reflected by the reflection mirror 19. . The excitation light reflected by the reflection mirror 19 passes through the dichroic mirror 31 and is irradiated on the sample S by the objective lens 23 through the imaging lens 21.

  Then, the CPU 58 controls the swinging operation of the scanners 15A and 15B of the observation scanning unit 15, whereby the excitation light is raster-scanned on the specimen S (image scan) as shown in FIG. Further, a scanning timing signal from the observation scanning waveform generation circuit 51 is sent to the image generation unit 57 via the CPU 58.

  When the fluorescent indicator is excited by irradiating the specimen S with the excitation light, and fluorescence is generated, the return light including the fluorescence is collected by the objective lens 23 and returns the optical path of the excitation light in the reverse direction. The return light passes through the imaging lens 21 and the dichroic mirror 31 and is reflected by the reflection mirror 19, then relayed by the relay lens 17, passes through the observation scanning unit 15, is reflected by the dichroic mirror 31, and is excited light. Branch from the optical path.

  The return light reflected by the dichroic mirror 31 is converted into light only by removing light other than fluorescence by the photometric filter 33 and is collected by the condenser lens 35. Of the fluorescence collected by the condenser lens 35, only the fluorescence generated from the focal position of the excitation light in the specimen S passes through the confocal pinhole 37 and is detected by the photoelectric conversion element 39. A luminance signal corresponding to the detected luminance of the fluorescence is sent to the image generation unit 57 by the photoelectric conversion element 39.

  In the image generation unit 57, the luminance signal sent from the photoelectric conversion element 39 is imaged (imaging) based on the scanning timing signal from the observation scanning unit 15 sent from the observation scanning waveform generation circuit 51. A two-dimensional image of the specimen S is generated. Thereby, the user can display the image on a monitor or the like and observe the specimen S.

  Next, when the specimen S is optically stimulated by the microscope system 100, first, stimulation light is generated from the stimulation light source 5. When the second acoustooptic element 41 is in the ON state under the control of the CPU 58, the stimulation light passes through the second acoustooptic element 41 along the optical path incident on the stimulation optical system 6, and the second acoustooptic element 41 is turned off. In the state, it passes through the second acoustooptic element 41 along the optical path that does not enter the stimulation optical system 6.

  When the second mechanical shutter 43 is open under the control of the CPU 58, the stimulus light that has passed through the second acoustooptic element 41 in the ON state passes through the optical path by being opened and enters the stimulus optical system 6. However, when the second mechanical shutter 43 is in the closed state, the stimulation light from the second acoustic element 41 is blocked by closing the optical path.

  Therefore, the CPU 58 can arbitrarily switch on / off the irradiation of the stimulation light to the specimen S in the second acoustooptic device 41 and the second mechanical shutter 43.

  The stimulus light that has passed through the second acoustooptic element 41 and the second mechanical shutter 43 is deflected by the stimulus scanning unit 45, relayed by the relay lens 47, reflected by the dichroic mirror 49, and then passed through the imaging lens 21. The sample S is irradiated by the objective lens 23.

  Then, the CPU 58 controls the swinging motion of the scanners 45A and 45B of the stimulus scanning unit 45, so that the stimulus light is scanned spirally in the photostimulation region of the specimen S as shown in FIG. scan). Thereby, the photostimulation area | region of the sample S can be photostimulated.

  In this case, the acoustooptic elements 11 and 41 can instantaneously switch between the ON state and the OFF state, but a weak leak light component of excitation light or stimulation light is generated regardless of the control of the diffraction angle. Therefore, it is not possible to completely switch between passing / blocking excitation light or stimulation light. On the other hand, the mechanical shutters 13 and 43 can completely switch between passing / blocking excitation light or stimulation light, but there is a time delay from the output of the drive signal to the actual opening / closing of the optical path.

  In the present embodiment, in the light stimulation of the sample S, for example, as shown in FIG. 6, the second mechanical shutter 43 is delayed before the second acoustooptic element 41 is switched from the OFF state to the ON state by the CPU 58. It starts to be switched from the closed state to the open state first by the amount of time.

  Thereby, even if the leakage light of the stimulation light is generated from the second acoustooptic element 41 in the OFF state until the timing when the sample S is irradiated with the stimulation light, the second mechanical shutter 43 closes the optical path even if the stimulation light leaks. Light can be blocked almost certainly. In addition, at the timing when the sample S is irradiated with the stimulation light, the second acoustooptic element 41 allows the stimulation light to pass through almost simultaneously with the switching while the second mechanical shutter 43 opens the optical path. Stimulation light can be irradiated.

  Further, the CPU 58 starts to switch the second mechanical shutter 43 from the open state to the closed state almost simultaneously with the switching of the second acousto-optic element 41 from the ON state to the OFF state.

Thereby, at the timing when the irradiation of the stimulation light to the specimen S is stopped, the second acousto-optic element 41 blocks the stimulation light almost simultaneously with the switching, so that the stimulation light can be blocked at a desired timing. In addition, after the stimulation light irradiation to the specimen S is stopped, the second mechanical shutter 43 closes the optical path even if the leakage light of the stimulation light is generated from the second acoustooptic element 41 in the OFF state. Therefore, the stimulation light can be blocked almost certainly.
Therefore, when a tornado scan is performed by the stimulation scanning unit 45, it is possible to prevent the stimulation light from being irradiated to an area other than the photostimulation area.

  Similarly, in the observation optical system 3, before the first acoustooptic device 11 is switched from the OFF state to the ON state, the first mechanical shutter 13 is switched from the closed state to the open state by the delay time. First, almost simultaneously with the first acoustooptic device 11 being switched from the ON state to the OFF state, the first mechanical shutter 13 starts to be switched from the open state to the closed state.

  Therefore, according to the microscope system 100 according to the present embodiment, the generation of leakage light by the acoustooptic elements 11 and 41 and the time delay of the mechanical shutters 13 and 43 are compensated for each other, and the sample S is applied at a timing other than the desired timing. It is possible to suppress the excitation light and the stimulation light from being irradiated, and to switch the irradiation of the excitation light and the stimulation light to the specimen S with high accuracy at an arbitrary timing.

  Further, the CPU 58 starts switching the mechanical shutters 13 and 43 to the open state first by the delay time before switching the acoustooptic elements 11 and 41 to the ON state, so that the excitation light or stimulation by the acoustooptic elements 11 and 41 is performed. The leakage light component of the acousto-optic elements 11 and 41 irradiated to the specimen S is minimized by minimizing the time difference between the switching of light passage / blocking and the switching of excitation light or stimulation light passing / blocking by the mechanical shutters 13 and 43. Can be less.

  FIG. 6 illustrates the case where the stimulus light is tornado-scanned on the specimen S by the stimulus optical system 6. For example, as shown in FIGS. 7 and 8, the CPU 58 uses the stimulus scanning unit 45 to scan the specimen. Stimulating light may be point-scanned multiple times at the same light stimulation position on S.

In this case, the CPU 58 makes the time for setting the second acoustooptic element 41 in the OFF state longer than twice the delay time of the second mechanical shutter 43, and the second acoustooptic element 41 is turned on / off. What is necessary is just to switch the open state / closed state of the 2nd mechanical shutter 43 repeatedly whenever it switches repeatedly.
By doing so, it is possible to intermittently irradiate the sample S with the stimulation light while suppressing the sample S from being irradiated with the stimulation light at a timing other than the desired timing.

  In this embodiment, for example, as shown in FIG. 9, when the light stimulation region is scanned by one line in the X direction by the X galvano scanner 45A (scanning period), the swing angle of the X galvano scanner 45A is restored. At the same time, the Y galvano scanner 45B is swung toward the next line in the Y direction (returning period) and scanned from the beginning of the next line in the X direction by the X galvano scanner 45A (scanning period). It may be scanned.

In this case, for example, as shown in FIG. 10, the second acousto-optic element 41 is turned on simultaneously with the start of the scanning period, and switched to the OFF state simultaneously with the end of the scanning period and the start of the retrace period. That's fine. The second mechanical shutter 43 starts to switch from the closed state to the open state for a delay time before the start of the scanning period of the first line, and starts to switch to the closed state at the end of the scanning period of the last line. do it.
By doing so, it is possible to minimize the irradiation of the stimulation light outside the photostimulation region in the specimen S.

This embodiment can be modified as follows.
In the present embodiment, before the second acoustooptic element 41 is switched to the ON state, the second mechanical shutter 43 starts to be switched to the open state first by the delay time, and the second acoustooptic element 41 is switched to the OFF state. At the same time, the second mechanical shutter 43 is started to be switched to the closed state. However, as a first modification, the second mechanical shutter 43 is opened before the image generation unit 57 starts imaging fluorescence. The second mechanical shutter 43 may start to be switched to the closed state at the same time when the fluorescence imaging is finished.

  In this case, as shown in FIG. 11, while the scanning of the excitation light is started by the observation scanning unit 15, the second mechanical shutter 43 starts to be switched from the closed state to the open state by the delay time first, and the scanning of the excitation light is finished. At the same time, the second mechanical shutter 43 may be switched from the open state to the closed state. Based on the frame signal from the CPU 58, the second acoustooptic device 41 is temporarily switched from the OFF state to the ON state while any one of the images is being acquired, and the sample S is irradiated with the stimulation light. And it is sufficient.

  By doing in this way, it is possible to almost certainly block the stimulation light by the second mechanical shutter 43 until the timing when the image generation unit 57 acquires the image. As a result, it is possible to minimize waste that the sample S is irradiated with the stimulation light while the image is not acquired.

  As a second modified example, as shown in FIG. 12, the microscope system 100 includes three (plural) stimulation light sources 5A, 5B, and 5C and stimulation light emitted from these stimulation light sources 5A, 5B, and 5C. A dichroic mirror (optical path combining unit) 61 that combines the optical paths, and three (a plurality of) second mechanical shutters 43A, 43B, and 43C disposed between the stimulation light sources 5A, 5B, and 5C and the dichroic mirror 61, It is good also as providing.

  In this case, the microscope system 100 reflects the stimulation light emitted from the stimulation light source 5B and the reflection mirror 63 that reflects the stimulation light emitted from the stimulation light source 5B. A dichroic mirror 65 that combines the optical path of the stimulating light from the stimulating light source 5B and the optical path of the stimulating light from the stimulating light source 5C so as to be incident on the dichroic mirror 61 by transmitting the stimulating light. Good.

The three stimulation light sources 5A, 5B, and 5C generate stimulation light having different wavelengths.
The dichroic mirror 61 transmits the stimulation light emitted from the stimulation light source 5A, while reflecting the stimulation light from the dichroic mirror 65, synthesizes the optical paths of these stimulation lights and enters the second acoustooptic device 41 . Be able to.

  By doing in this way, passage / blocking of stimulation light can be reliably switched for each of the stimulation light sources 5A, 5B, 5C by the second mechanical shutters 43A, 43B, 43C. The stimulus light is reliably blocked by the second mechanical shutter 43A (43B, 43C) except for the timing at which the sample S is irradiated with the stimulus light from any one of the stimulus light sources 5A (5B, 5C). At the timing when the sample S is irradiated with the stimulation light from (5B, 5C), the second acoustooptic device 41 can pass the stimulation light at a desired timing.

[Second Embodiment]
Next, a microscope system according to the second embodiment of the present invention will be described.
As shown in FIG. 13, the microscope system 200 according to the present embodiment replaces the stimulation light source 5 and the second acoustooptic device 41, and a laser diode (laser light source) capable of adjusting the intensity of the laser light according to the drive voltage. 105, and the CPU 58 is different from the first embodiment in that the laser diode 105 and the second mechanical shutter 43 control ON / OFF of the laser light irradiation to the specimen S.
In the following, portions having the same configuration as those of the microscope system 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  The laser diode 105 can be switched between an ON state in which laser light is generated and an OFF state in which laser light is not generated. Laser light emitted from the laser diode 105 enters the stimulation optical system 6 via the second mechanical shutter 43.

  The laser diode 105 can be switched instantaneously between an ON state and an OFF state. However, even in the OFF state, a weak leakage light component of the laser light is generated, so that the laser light generation timing is completely switched. I can't.

Instead of the second control circuit 54, the control device 7 includes a control circuit (not shown) that outputs a control signal for switching the laser diode 105 between the ON state and the OFF state.
The CPU 58 generates a control signal from the control circuit and controls switching of the laser diode 105 between the ON state and the OFF state.

  Further, the CPU 58 starts to switch the second mechanical shutter 43 to the open state first by the delay time before switching the laser diode 105 to the ON state, and simultaneously switches the laser diode 105 to the OFF state and closes the second mechanical shutter 43. It is also possible to start switching to.

The operation of the microscope system 200 configured as described above will be described.
The case of observing the specimen S with the excitation light source 2 and the observation optical system 3 is the same as that in the first embodiment, and thus the description thereof is omitted.
When the sample S is optically stimulated by the microscope system 200, first, the CPU 58 switches the laser diode 105 to the ON state to generate laser light.

  When the second mechanical shutter 43 is in the open state under the control of the CPU 58, the laser light emitted from the laser diode 105 is incident on the stimulation optical system 6 through the optical path by being opened. When the shutter 43 is in the closed state, the laser beam from the laser diode 105 is blocked by closing the optical path.

When the laser diode 105 is switched to the OFF state by the CPU 58, the generation of laser light is stopped.
Therefore, the CPU 58 can arbitrarily switch ON / OFF the irradiation of the sample S with the laser light in the laser diode 105 and the second mechanical shutter 43.

  In this embodiment, in the light stimulation of the sample S, the second mechanical shutter 43 is switched from the closed state to the open state first by the delay time before the laser diode 105 is switched from the OFF state to the ON state by the CPU 58. Begin to be caught.

  Therefore, until the timing at which the sample S is irradiated with the laser beam, even if laser beam leakage light is generated from the laser diode 105 in the OFF state, the laser beam is almost certainly blocked by the second mechanical shutter 43 blocking the optical path. can do. Further, at the timing of irradiating the sample S with laser light, the laser diode 105 generates laser light almost simultaneously with switching while the second mechanical shutter 43 opens the optical path, so that laser light is applied to the sample S at a desired timing. Can be irradiated.

  Further, the CPU 58 starts to switch the second mechanical shutter 43 from the open state to the closed state almost simultaneously with switching of the laser diode 105 from the ON state to the OFF state.

  Thereby, at the timing when the irradiation of the laser beam to the specimen S is stopped, the laser diode 105 stops generating the laser beam almost simultaneously with the switching, so that the laser beam can be shut off at a desired timing. Further, after the irradiation of the laser beam to the specimen S is stopped, the second mechanical shutter 43 closes the optical path even if the laser beam leaks from the laser diode 105 in the OFF state. Can be blocked almost certainly.

  Therefore, according to the microscope system 200 according to the present embodiment, the generation of leakage light from the laser diode 105 and the time delay of the second mechanical shutter 43 are mutually compensated, and the laser light is applied to the sample S at a timing other than the desired timing. Can be suppressed, and ON / OFF of laser light irradiation to the specimen S can be switched at an arbitrary timing.

  In the present embodiment, the case where the laser diode 105 is employed instead of the stimulation light source 5 and the second acoustooptic element 41 is illustrated, but a laser diode (laser light source) is replaced instead of the excitation light source 2 and the first acoustooptic element 11. It may be adopted.

  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, the present invention is not limited to those applied to the above-described embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. .

  In the first embodiment, the mechanical shutters 13 and 43 are switched before the acoustooptic elements 11 and 41 by a delay time. However, before the acoustooptic elements 11 and 41 are switched, the mechanical shutters 13 and 43 are switched. By switching, it is only necessary to compensate for the occurrence of leaked light by the reverberating optical elements 11 and 41 and the time delay of the mechanical shutters 13 and 43, and the timing for switching the mechanical shutters 13 and 43 may be slightly earlier than the delay time. It may be late.

  Also in the second embodiment, the second mechanical shutter 43 is switched before the laser diode 105 by a delay time. However, by switching the second mechanical shutter 43 before switching the laser diode 105, the laser diode is switched. The generation of the leaked light from 105 and the time delay of the second mechanical shutter 43 may be compensated for each other, and the timing for switching the second mechanical shutter 43 may be slightly earlier or later than the delay time. The same applies to the case where a laser diode (laser light source) is employed instead of the excitation light source 2 and the first acoustooptic device 11.

  Moreover, in each said embodiment, although the structure arrange | positioned in order of the 1st acousto-optic element 11 and the 1st mechanical shutter 13 from the excitation light source 2 side was illustrated and demonstrated, it replaced with this and the 1st from the excitation light source 2 side was demonstrated. One mechanical shutter 13 and the first acoustooptic device 11 may be arranged in this order. Similarly, in the first embodiment, the configuration in which the second acoustooptic element 41 and the second mechanical shutter 43 are arranged in this order from the stimulus light source 5 side has been described as an example. It is good also as arrange | positioning in order of 2 mechanical shutter 43 and the 2nd acoustooptic device 41. FIG.

2 Observation light source (light source)
3 Observation optical system (irradiation optical system)
5 Stimulation light source (light source)
6 Stimulation optical system (irradiation optical system)
DESCRIPTION OF SYMBOLS 11 1st acoustooptic element 13 1st mechanical shutter 41 2nd acoustooptic element 43 2nd mechanical shutter 57 Image generation part (image acquisition part)
58 CPU (control unit)
61 Dichroic mirror (Optical path synthesis unit)
100,200 Microscope system 105 Laser diode (laser light source)

Claims (10)

A light source that generates light;
An irradiation optical system for irradiating the specimen with light emitted from the light source;
To light emitted from the light source, the acousto-optic device which can switch on and the OFF state you angle does not enter the ON state you angle incident on the irradiation optical system,
A mechanical shutter that is detachably provided in an optical path between the light source and the irradiation optical system, and that can be switched between an open state that opens the optical path and a closed state that closes the optical path;
A control unit for controlling ON / OFF of light irradiation to the specimen by the acoustooptic device and the mechanical shutter;
A microscope system in which the control unit switches the mechanical shutter to an open state before switching the acoustooptic device to an ON state.   The control unit opens the mechanical shutter before the acousto-optic element by a time delay required for the mechanical shutter to be detached from the optical path after inputting the insertion / removal operation signal to the mechanical shutter. The microscope system according to claim 1, wherein the microscope system starts to be switched to. Wherein the control unit, the acousto-optic element at the same time as the switch to OFF state, the microscope system according to the mechanical shutter to claim 1 or claim 2 starts switching to the closed state. The controller makes the acousto-optic element OFF state longer than twice the delay time of the mechanical shutter, and sets the acousto-optic element ON state / off state and the mechanical shutter open state / closed state. The microscope system according to claim 2 or 3, wherein the microscope system is repeatedly switched. A plurality of the light sources that generate light of different wavelengths;
An optical path synthesis unit that synthesizes an optical path with light emitted from the plurality of light sources,
A plurality of mechanical shutters disposed between each of the light sources and the optical path combining unit;
The microscope system according to any one of claims 1 to 4, wherein the acoustooptic device is disposed on an optical path of light synthesized by the optical path synthesis unit. A laser light source that adjusts the intensity of the laser light in accordance with the drive voltage and can be switched between an ON state in which the laser light is generated and an OFF state in which the laser light is not generated;
An irradiation optical system for irradiating the sample with laser light emitted from the laser light source;
A mechanical shutter that is detachably provided in an optical path between the laser light source and the illumination optical system, and that can be switched between an open state that opens the optical path and a closed state that closes the optical path;
A controller that controls ON / OFF of laser light irradiation to the specimen by the laser light source and the mechanical shutter;
A microscope system in which the control unit switches the mechanical shutter to an open state before switching the laser light source to an ON state.   The control unit opens the mechanical shutter before the laser light source by the time delay required for the mechanical shutter to be detached from the optical path after inputting the signal for insertion / removal operation to the mechanical shutter. The microscope system according to claim 6, wherein switching is started. The microscope system according to the control unit, the laser light source at the same and to switch to the OFF state, the claim of the mechanical shutter starts switching to the closed state 6 or claim 7. The said control part makes the OFF state of the said laser light source longer than the time twice the delay time of the said mechanical shutter, and switches the ON state / OFF state of this laser light source repeatedly. Microscope system. An image acquisition unit that images the return light from the sample and acquires the image of the sample;
The microscope system according to claim 1 or 9, wherein the control unit switches the mechanical shutter to an open state before the imaging unit starts imaging the return light.

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