JP5176521B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a laser scanning microscope such as a confocal laser scanning microscope or a confocal fluorescent laser scanning microscope.

    There has been proposed a laser scanning microscope in which an optical system for light stimulation is combined with an optical system for imaging (see, for example, Patent Document 1). According to this microscope, it is possible to give a stimulus to a part of a specimen with light of a specific wavelength and observe a change occurring in the vicinity (light stimulus observation).

A plurality of scanning optical systems and one detection optical system are provided, and the detection optical systems are optically coupled to the specimen via one scanning optical unit of the plurality of scanning optical systems (see Patent Document 2). ). Accordingly, the fluorescence generated from the fluorescent reagent excited by the first light beam travels in the opposite direction along the same optical path as the first light beam and is guided to the detection optical system. In addition, the fluorescence generated from the fluorescent reagent excited by the second light beam travels in the opposite direction along the same optical path as the first light beam and is guided to the detection optical system.
Japanese Patent Laid-Open No. 10-206742 Japanese Patent Laid-Open No. 2005-189290

  However, in the microscope described in Patent Document 1, since the optical system for light stimulation is independent of the optical system for imaging, both the light source and the galvano scanner arranged in the optical system for light stimulation are light-stimulated. Can only be used for

  For example, even if the wavelength for imaging and the wavelength for photostimulation of a specimen are the same, it is necessary to mount one light source on both the imaging optical system and the photostimulation optical system. There is. In the microscope described in Patent Document 2, the fluorescence generated from the fluorescent reagent excited by the second light beam is different from the second optical scanning unit (scanner) used for scanning the second light beam. Since they are guided to the detection optical system using a unit (scanner), both need to be of the same type, and in order to acquire an image, it is necessary to synchronize with high accuracy.

  Accordingly, an object of the present invention is to provide a laser scanning microscope that can increase the degree of freedom of observation while keeping the configuration simple.

The laser scanning microscope of the present invention is configured to guide light from a light source unit to a surface to be observed and to guide light from the surface to be observed to a detector, between the light path dividing unit and the surface to be observed. And an optical path switching means for separating the optical path between the optical path dividing means and the surface to be observed between a first optical path and a second optical path having different paths, and a first optical path disposed in the first optical path. 1 scanner and a second scanner disposed in the second optical path and driven independently of the first scanner, and the optical path switching means includes the first optical path and the second optical path. The first optical path switching unit includes a first optical path switching unit disposed at one branch location and a second optical path switching unit disposed at the other branch location of the first optical path and the second optical path. Between the unit and the first scanner, and the first optical path switch. And further comprising a plurality of filters placed respectively between the unit and the second scanner.

  ADVANTAGE OF THE INVENTION According to this invention, the laser scanning microscope which can raise the freedom degree of observation is suppressed, suppressing a structure simply.

[First Embodiment]
Hereinafter, the first embodiment will be described. This embodiment is an embodiment of a fluorescent confocal laser scanning microscope system.

  First, the configuration of this system will be described.

  FIG. 1 is a configuration diagram of the present system. As shown in FIG. 1, this system includes a microscope main body 100, a controller 20, a computer 21, a monitor 22, an input device 23, and the like.

  The microscope body 100 includes a laser unit 1, an optical fiber 7, a collimating lens 8, a dichroic mirror 9, an optical path switching unit 10, a control type galvano scanner 11, an optical path switching unit 13, a resonance type galvano scanner 12, a relay lens 14, and an objective lens. 15, a specimen 16, a condensing lens 17, a pinhole diaphragm 18 for confocal detection, a photodetector 19, and the like are arranged. Among these, the dichroic mirror 9, the condensing lens 17, the pinhole aperture 18, and the photodetector 19 constitute a detection optical system 100A. Note that the specimen 16 is a specimen for fluorescence observation (a specimen to which a fluorescent dye is added) supported on a stage (not shown).

  The control type galvano scanner 11 includes a main scanning galvanometer mirror and a sub-scanning galvanometer mirror arranged in series, and the resonance type galvano scanner 12 is a main scanning resonance galvanometer mirror arranged in series. And a sub-scanning control type galvanometer mirror. Among these, the control type galvano scanner 11 has an advantage that the scan area can be freely set although the scan speed is slow. Therefore, the irradiation destination of the laser beam is limited to a desired partial area of the observation area of the specimen 16. The resonance-type galvano scanner 12 is effective when the specimen 16 is laser-scanned at a high speed because the resonant galvano scanner 12 has an advantage that the scanning speed is fast. is there.

  The laser unit 1 is equipped with a plurality of types of laser light sources (here, two laser light sources 2 and 3). The outgoing optical paths of the laser light sources 2 and 3 are integrated into a common optical path by a combiner mirror 4, and an acousto-optic filter (AOTF) 5 is inserted in the common optical path. By controlling the AOTF 5 and the individual laser light sources 2 and 3, the laser unit 1 performs setting of a light source to be used, on / off of emitted light, adjustment of intensity of emitted light, and the like.

  Laser light emitted from the laser unit 1 enters one end of the optical fiber 7 via the fiber coupler 6. The laser light propagates through the optical fiber 7, exits from the other end of the optical fiber 7, is collimated into a collimated lens 8, and then enters the dichroic mirror 9. The laser light passes through the dichroic mirror 9 and enters the dichroic mirror 10D of the optical path switching unit 10.

  The short-wavelength laser light transmitted through the dichroic mirror 10D passes through the first filter 10E, passes through the optical path R1, is reflected by the control type galvano scanner 11, and then enters the dichroic mirror 13D of the optical path switching unit 13. Since the characteristics of the dichroic mirror 13D are set to be the same as the characteristics of the dichroic mirror 10D, the laser light passing through the optical path R1 is transmitted through the dichroic mirror 13D, passes through the relay lens 14 and the objective lens 15, and is on the sample 16. To form a spot. The fluorescence generated at the spot (having a wavelength slightly longer than that of the laser beam) travels in the same optical path R1 as the laser beam forming the spot, and is reflected by the control type galvano scanner 11 and enters the first filter 10E. The first filter 10E has a characteristic of transmitting a laser beam having a short wavelength and not transmitting a longer wavelength, and the fluorescence is shielded here. When the control type galvano scanner 11 is driven in this state, the spot scans the sample 16 two-dimensionally.

  On the other hand, the laser beam having a relatively long wavelength reflected from the dichroic mirror 10D passes through the optical path R2 different from the optical path R1, passes through the second filter 10F, and is reflected by the resonant galvano scanner 12, and then the optical path switching unit 13 Is incident on the dichroic mirror 13D. Since the characteristics of the dichroic mirror 13D are set to be the same as those of the dichroic mirror 10D, the laser light passing through the optical path R2 is reflected by the dichroic mirror 13D, passes through the relay lens 14 and the objective lens 15, and is spotted on the sample 16. Form. The fluorescence generated at the spot portion (having a wavelength slightly longer than that of the laser beam that forms the spot) follows the same optical path R2 as the laser beam that forms the spot, and is reflected by the resonant galvano scanner 12 to be reflected by the second laser beam. The light passes through the filter 10F, reflects off the dichroic mirror 10D, and travels toward the dichroic mirror 9. When the resonant galvano scanner 12 is driven in this state, the spot scans the sample 16 two-dimensionally.

  The fluorescence incident on the dichroic mirror 9 is reflected by the dichroic mirror 9 and taken into the detection optical system 100A. The fluorescent light collected by the condenser lens 17 and passed through the pinhole diaphragm 18 enters the photodetector 19 and is converted into an electrical signal. The optical path switching unit 10 is equipped with a dichroic mirror 10D, a total reflection mirror 10M, and a hollow block (not shown) and is inserted into the optical path.

  Here, the configuration of the optical path switching units 10 and 13 will be described.

  FIGS. 9A and 9B are diagrams illustrating the configuration of the optical path switching units 10 and 13.

  Each of the optical path switching units 10 and 13 includes a turret. The turrets 50 and 51 are each configured with four hollow blocks 10R and 13R every 90 degrees in the circumferential direction, and a maximum of four optical elements can be arranged on these. Dichroic mirrors 10D and 13D and total reflection mirrors 10M and 13M are respectively arranged in the two hollow blocks 10R and 13R, and the remaining two hollow blocks 10R and 13R are used in an empty state. However, one of them may be a dichroic mirror having characteristics different from those of the dichroic mirrors 10D and 13D.

  Further, the turrets 50 and 51 are respectively formed with gears 50H and 51H having the same number of teeth on the outer periphery, and the turrets 50 and 51 are arranged adjacent to each other so that the gears mesh with each other. The small gear 52 is adjacently disposed so that the gear meshes with the gear 50 </ b> H of the turret 50, and the rotation shaft of the small gear 52 is driven by the motor 53.

  Therefore, as the small gear 52 driven by the motor 53 rotates, the turrets 50 and 51 rotate in conjunction with each other (the turrets 50 and 51 are each provided with a rotation shaft (not shown). The motor 53 and the bearings 54 and 55 are fixed on the base 56 with screws, and the dichroic mirror 10D is disposed in the optical path. A dichroic mirror 13D is disposed in the optical path. Similarly, when the total reflection mirror 10M is disposed in the optical path, the total reflection mirror 13M is disposed in the optical path.

  The first filter 10E and the second filter 10F are held by holding members 61 and 62 connected to the turret 50, and are arranged so that an angle formed with the dichroic mirror 10D is about 33 degrees before and after the dichroic mirror 10D. Has been. Therefore, these are inserted into the optical path simultaneously with the switching of the dichroic mirror 10D, and the incident angle is arranged at about 12 degrees with respect to each optical path.

  Note that the first filter 10E and the second filter 10F are preferably arranged at an incident angle other than 0 degrees with respect to each optical path. This is because if these are arranged at an incident angle of 0 degree with respect to each optical path, the light that has not been transmitted is reflected and returns to the optical path, causing flare.

  By adopting such a configuration, the two turrets 50 and 51 can be controlled in conjunction with only one motor 53, and space saving and cost reduction are realized.

    As shown in FIG. 1, when the setting element by the optical path switching units 10 and 13 is set to a combination of the dichroic mirrors 10D and 13D, the optical path between the dichroic mirror 9 and the relay lens 14 is the optical path R1 for each wavelength. It is separated from the optical path R2.

  On the other hand, when the setting element by the optical path switching units 10 and 13 is set to the combination of the total reflection mirrors 10M and 13M, the optical path between the dichroic mirror 9 and the relay lens 14 is only the optical path R2 regardless of the wavelength.

  When the setting elements by the optical path switching units 10 and 13 are set in a combination of hollow blocks, the optical path between the dichroic mirror 9 and the relay lens 14 is only the optical path R1 regardless of the wavelength.

  Further, the optical path switching units 10 and 13 and other driving units (the laser unit 1, the control type galvano scanner 11, the resonance type galvano scanner 12, the photodetector 19, etc.) of the microscope main body 100 are controlled by the controller 20. . The controller 20 is under the control of the computer 21, and an instruction from the user is given to the computer 21 via the monitor 22 and the input device 23, and is given to the controller 20 via the computer 21.

  The controller 20 gives necessary instructions and drive signals to each drive unit of the microscope body 100 in accordance with instructions from the computer 21, thereby setting and driving the microscope body 100.

  For example, the controller 20 sets the microscope main body 100 so that the optical path R2 is effective, and then sends an electrical signal from the photodetector 19 while synchronously driving the laser unit 1, the resonant galvano scanner 12, and the photodetector 19. If captured, fluorescence image data of the observation region of the specimen 16 can be acquired (imaging). This fluorescent image data is sent from the controller 20 to the computer 21, sent to the monitor 22 as needed, or stored in the computer 22.

  As described above, the microscope main body 100 of the present system allows the pair of galvano scanners (the control galvano scanner 11 and the resonance galvano scanner 12) to be connected between the dichroic mirror 9 and the specimen 16 by using the optical path switching units 10 and 13. Since they are arranged in parallel to the optical path, one light source unit 1 and one detection optical system 100A are shared by a pair of galvano scanners (control galvano scanner 11 and resonant galvano scanner 12). Will be.

  Therefore, although the microscope main body 100 of this system is equipped with only one laser unit 1 and one detection optical system 100A, the projecting destination of the laser unit 1 is set between a pair of galvano scanners. It is possible to select a galvano scanner used for imaging or a pair of galvano scanners.

  The optical path switching units 10 and 13 include a beam splitter (in this case, dichroic mirrors 10D and 13D) as one of optical elements that can be inserted into the optical path, so that the laser light from the laser unit is paired with a galvano scanner (control). It is also possible to project simultaneously on the galvano scanner 11 and the resonant galvano scanner 12).

  In the microscope main body 100 of this system, the branch point of the optical paths R1 and R2 where the pair of galvano scanners are arranged is between the dichroic mirror 9 and the relay lens 14, but the relay lens 4 and the objective lens 15 Or in the optical path of the relay lens 4. However, when one of the branch points is in the optical path of the relay lens 4, it is necessary to dispose a part of the relay lens in each of the optical paths R1 and R2, so that the number of optical elements is slightly increased.

  In the microscope main body 100 of the present system, the optical path switching units 10 and 13 are configured by turrets, but may be configured by other mechanisms such as a slide mechanism.

  In the microscope main body 100 of the present system, the optical path switching units 10 and 13 are motorized by a motor. However, the motor may be omitted and the switching may be performed manually.

[Second Embodiment]
The second embodiment will be described below. The present embodiment is an embodiment of a light stimulus observation method that performs light stimulus and imaging simultaneously using the system of the first embodiment.

  Here, the excitation wavelength of the fluorescent dye applied to the specimen 16 is 488 nm, and the wavelength of the light stimulus to be given to the specimen 16 is 405 nm.

In this case, each part of the microscope main body 100 is set as follows, for example.
Laser light source 2: UV laser light source (wavelength 405 nm)
Laser light source 3: Argon laser light source (wavelength 488 nm)
Dichroic mirror 9: Transmits light on the short wavelength side including 488 nm and reflects light on the longer wavelength side than 488 nm. Dichroic mirror 10D, 13D: Transmits light in the vicinity of 405 nm (width of about 10 nm). Reflecting light on the long wavelength side including 488 nm First filter 10E: Transmitting light in the vicinity of 405 nm (width of about 10 nm) and not transmitting light on the longer wavelength side Second filter 10F: Transmitting light on the long wavelength side including 488 nm and not transmitting light in the vicinity of 405 nm (width of about 10 nm) The procedure of light stimulus observation is as shown in FIG.

(Step 1)
The controller 20 sets the use light source of the laser unit 1 to the laser light source 3 (argon laser light source), and sets the setting elements of the optical path switching units 10 and 13 to a combination of the dichroic mirrors 10D and 13D as shown in FIG. To do. At the same time, the first filter 10E and the second filter 10F are also inserted into the optical paths R1 and R2.

  At this time, laser light that can be emitted from the laser unit 1 is only argon laser light having a wavelength of 488 nm. As shown by a solid line in FIG. 3, most of the argon laser light can reach the specimen 16 through the optical path R <b> 2, and the spot of the argon laser light can be detected by the resonant galvano scanner 12 in the entire observation region ( (See the lower part of FIG. 3). Fluorescence generated at the spot can be reflected by the dichroic mirror 9 as indicated by a dotted line in FIG. 3, and thus is extracted to the detection optical system 100A side.

  Note that, as indicated by the alternate long and short dash line in FIG. 3, the argon laser light that leaks and passes through the dichroic mirror 10 </ b> D without being reflected does not reach the sample 16 because it is reflected by the first filter 10 </ b> E. Therefore, it is possible to prevent the argon laser beam from being scanned unnecessarily on the specimen by the control type galvano scanner 11.

(Step 2)
The controller 20 drives the laser unit 1, the resonant galvano scanner 12, and the photodetector 19, and performs imaging of the entire observation region (see the lower part of FIG. 3) of the specimen 16 with argon laser light. The fluorescence image data of the specimen 16 acquired by this imaging is sent to the computer 21.

(Step 3)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22 and causes the user to designate one or a plurality of partial areas on which light stimulation should be performed on the screen. The user operates the input device 23 and designates a desired partial area to the computer 21. Information on the partial area designated by the user is sent to the controller 20.

(Step 4)
The controller 20 sets the scan area of the control type galvano scanner 11 to one or a plurality of partial areas designated by the user, and uses both the laser light sources 2 and 3 as the light source of the laser unit 1 (the ultraviolet laser light source and the argon light source). Laser light source).

  At this time, laser light that can be emitted from the laser unit 1 is both argon laser light having a wavelength of 488 nm and ultraviolet laser light having a wavelength of 405 nm.

    As in Step 1, the argon laser light can reach most of the specimen 16 through the optical path R2 as indicated by the solid line in FIG. 3, and the argon laser light spot is obtained by the resonant galvano scanner 12. It is possible to scan the entire observation area of the specimen 16 (see the lower part of FIG. 3). Fluorescence generated at the spot can be reflected by the dichroic mirror 9 as indicated by a dotted line in FIG. 3, and thus is extracted to the detection optical system 100A side.

  Note that, as indicated by the alternate long and short dash line in FIG. 3, the argon laser light that leaks and passes through the dichroic mirror 10 </ b> D without being reflected does not reach the sample 16 because it is reflected by the first filter 10 </ b> E. Therefore, it is possible to prevent the argon laser beam from being scanned unnecessarily on the specimen by the control type galvano scanner 11.

  On the other hand, as indicated by a solid line in FIG. 4, the ultraviolet laser light can reach the specimen 16 through the optical path R1, and the spot of the ultraviolet laser light is caused by the control type galvano scanner 11 to be a partial region of the specimen 16 (lower part of FIG. 4). Scan).

  Note that, as indicated by the alternate long and short dash line in FIG. 4, the ultraviolet laser light that leaks and reflects without passing through the dichroic mirror 10 </ b> D is reflected by the second filter 10 </ b> F and thus does not reach the sample 16. Therefore, it is possible to prevent the ultraviolet laser beam from being unnecessarily scanned on the specimen by the resonance type galvano scanner 12.

  A part of fluorescence (which is considered to be generated somewhat) in the spot of the ultraviolet laser beam is reflected by the dichroic mirror 13D and the rest is transmitted, as indicated by a dotted line and a two-dot chain line in FIG. Since the fluorescence reflected by the dichroic mirror 13D is incident on the resonance type galvano scanner 12, it does not pass through the same scanner and therefore does not descan and does not pass through the pinhole of the detection optical system 100A. Further, since the fluorescence transmitted through the dichroic mirror 13D is descanned by the control type galvano scanner 11 and reflected by the first filter 10E, it does not pass through the pinhole of the detection optical system 100A. Therefore, it is possible to prevent the fluorescence generated from the spot of the ultraviolet laser light from being detected by the photodetector 19 by being mixed with the fluorescence generated from the spot of the argon laser light.

(Step 5)
The controller 20 drives the laser unit 1, the control type galvano scanner 11, the resonance type galvano scanner 12, and the photodetector 19, and applies optical stimulation to a partial region of the specimen 16 (see the lower part of FIG. 4) with ultraviolet laser light. Then, imaging of the entire observation region (see the lower part of FIG. 3) of the specimen 16 is performed with argon laser light. The fluorescence image data of the specimen 16 acquired by this imaging is sent to the computer 21.

(Step 6)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22. On the screen, the user can observe the state of the specimen 16 when light stimulation is performed. The computer 21 stores the fluorescence image data as necessary. (End of step 6).

  Each step described above is repeated or returned to the previous step as necessary.

  As described above, in the present embodiment, since the system of the first embodiment, in particular, the dichroic mirrors 10D and 13D of the optical path switching units 10 and 13 are used effectively, light stimulation and imaging can be performed simultaneously.

  In step 5 described above, light stimulation (driving the laser light source 2 and the control type galvano scanner 11) and imaging (driving the laser light source 3, the resonance type galvano scanner 12, and the light detector 19) are performed only once. There were no, but these are not the only cases. For example, the light stimulation may be repeated continuously. Then you can stimulate more strongly. Further, imaging may be repeated continuously. By doing so, fluorescent image data for a plurality of consecutive frames can be obtained, and thus it is possible to observe the temporal change of the specimen 16 immediately after the light stimulation. In addition, the number of times of light stimulation may be specified by the user while performing the light stimulation, imaging, and monitor output in Step 5 and Step 6 described above.

  In Step 1 described above, the setting element of the optical path switching units 10 and 13 is a combination of dichroic mirrors 10D and 13D, but may be a combination of total reflection mirrors 10M and 13M. In this case, however, it is necessary to change the setting elements of the optical path switching units 10 and 13 to a combination of the dichroic mirrors 10D and 13D in Step 4.

[Third Embodiment]
Hereinafter, a third embodiment will be described. This embodiment is an embodiment of a light stimulus observation method that uses a common light source for light stimulus and imaging using the system of the first embodiment.

  Here, the excitation wavelength of the fluorescent dye applied to the specimen 16 is 488 nm, and the wavelength of the light stimulus to be applied to the specimen 16 is 488 nm.

In this case, the microscope main body 100 is set as follows, for example.
Laser light source 3: Argon laser light source (wavelength 488 nm)
Dichroic mirror 9: Transmits light on the short wavelength side including 488 nm and reflects light on the longer wavelength side than 488 nm. In this embodiment, the laser light source 3 and the dichroic mirrors 10D and 13D are not used. These settings are arbitrary. Therefore, the setting of the microscope main body 100 may be the same as that of the second embodiment.

  The procedure of light stimulus observation is as shown in FIG.

(Step 1)
The controller 20 sets the use light source of the laser unit 1 to the laser light source 3 (argon laser light source), and sets the setting elements of the optical path switching units 10 and 13 to a combination of total reflection mirrors 10M and 13M as shown in FIG. Set. At this time, the intensity of the emitted light from the laser unit 1 is set to an imaging intensity (low intensity).

  At this time, laser light that can be emitted from the laser unit 1 is only low-intensity argon laser light. The low-intensity argon laser light can reach the specimen 16 through the optical path R2 as indicated by a solid line in FIG. 6, and the spot of the argon laser light is detected by the resonant galvano scanner 12 over the entire observation region ( (See the lower part of FIG. 6). Since the fluorescence generated at the spot can be reflected by the dichroic mirror 9 as shown by the dotted line in FIG. 6, it is extracted to the detection optical system 100A side.

(Step 2)
The controller 20 drives the laser unit 1, the resonant galvano scanner 12, and the photodetector 19, and performs imaging of the entire observation region (see the lower part of FIG. 6) of the specimen 16 with low-intensity argon laser light. The fluorescence image data of the specimen 16 acquired by this imaging is sent to the computer 21.

(Step 3)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22 and causes the user to designate one or a plurality of partial areas on which light stimulation should be performed on the screen. The user operates the input device 23 and designates a desired partial area to the computer 21. Information on the partial area designated by the user is sent to the controller 20. (Step 4)
The controller 20 sets the scan area of the control type galvano scanner 11 to one or a plurality of partial areas designated by the user. Further, the controller 20 changes the intensity of the emitted light of the laser unit 1 to the intensity for light stimulation (high intensity), and the setting elements of the optical path switching units 10 and 13 are hollow blocks 10B as shown in FIG. , 13B.

  At this time, laser light that can be emitted from the laser unit 1 is only high-intensity argon laser light. As shown by the solid line in FIG. 7, the high-intensity argon laser light can reach the sample 16 through the optical path R1, and the spot of the argon laser light is controlled by the control-type galvano scanner 11 in a partial region (FIG. 7 (see lower part)). Since the fluorescence generated at the spot can be reflected by the dichroic mirror 9 as shown by a dotted line in FIG. 7, it is extracted to the detection optical system 100A side.

(Step 5)
The controller 20 drives the laser unit 1 and the control type galvano scanner 11 to give a light stimulus to a partial region of the specimen 16 (see the lower part of FIG. 7) with a high-intensity argon laser beam. At this time, there is no need for imaging, and there is no need to drive the photodetector 19.

(Step 6)
The controller 20 changes the intensity of the emitted light of the laser unit 1 to the intensity for imaging (low intensity), and the setting elements of the optical path switching units 10 and 13 are totally reflected mirrors 10M and 13M as shown in FIG. Change to the combination.

  At this time, laser light that can be emitted from the laser unit 1 is only low-intensity argon laser light. The low-intensity argon laser light can reach the specimen 16 through the optical path R2 as indicated by a solid line in FIG. 6, and the spot of the argon laser light is detected by the resonant galvano scanner 12 over the entire observation region ( (See the lower part of FIG. 6). Since the fluorescence generated at the spot can be reflected by the dichroic mirror 9 as shown by the dotted line in FIG. 6, it is extracted to the detection optical system 100A side.

(Step 7)
The controller 20 drives the laser unit 1, the resonant galvano scanner 12, and the photodetector 19, and performs imaging of the entire observation region (see the lower part of FIG. 6) of the specimen 16 with low-intensity argon laser light. The fluorescence image data of the specimen 16 acquired by this imaging is sent to the computer 21.

(Step 8)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22. On the screen, the user can observe the state of the specimen 16 immediately after the light stimulus. The computer 21 stores the fluorescence image data as necessary (step 8).

  Each step described above is repeated or returned to the previous step as necessary.

  As described above, in this embodiment, since the hollow blocks 10B and 13B and the total reflection mirrors 10M and 13M of the optical path switching units 10 and 13 are used effectively, it is common to the optical stimulation and imaging. A light source can be used.

  In step 5 described above, the light stimulation is performed only once, but may be performed repeatedly in succession. Then you can stimulate more strongly.

  Note that in step 7 described above, imaging was performed only once, but may be performed repeatedly in succession. By doing so, fluorescent image data for a plurality of consecutive frames can be obtained, and thus it is possible to observe the temporal change of the specimen 16 immediately after the light stimulation.

[Others]
In the above-described microscope body 100, two types of light sources are mounted on the light source unit 1, but three or more types may be used. A visible laser (wavelength 430 nm or the like) may be used for the stimulation laser light source 3. In addition to the ultraviolet laser light source and the argon laser light source, an IR laser light source (for example, wavelength 710 nm) used for two-photon stimulation, a wavelength variable IR laser light source (for example, wavelength 760 nm to 960 nm), and the like may be mounted. Further, stimulation and imaging may be performed with two photons by using a plurality of IR laser light sources or an IR laser light source that simultaneously outputs a plurality of wavelengths.

  In addition, in the dichroic mirrors 10D and 13D, generally, the wavelength characteristic (reflectance) is more flat in the reflection band than in the transmission band in the entire band. Therefore, the imaging wavelength can be obtained by transmitting the laser light for light stimulation (short wavelength) and reflecting the imaging laser light and the fluorescence (long wavelength) generated thereby as in the microscope main body 100 described above. It becomes easy to flatten the characteristics of the band dichroic mirrors 10D and 13D.

  Thus, by setting the wavelength characteristics of the dichroic mirrors 10D and 13D to a reflectance of 95% or more from 488 nm to 560 nm, it is possible to perform imaging of fluorescent dyes excited with an argon laser light source (wavelength 488 nm) with high image quality.

  More desirably, by setting the reflectivity to 95% or more from 488 nm to 620 nm, imaging of fluorescent dyes excited by not only an argon laser light source (wavelength 488 nm) but also a longer wavelength helium neon laser light source (wavelength 543.5 nm) Can be done with high image quality.

More desirably, by setting the reflectivity to 95% or higher from 488 nm to 750 nm, it is possible to perform imaging of a fluorescent dye excited by an LD light source having a longer wavelength (wavelength 640 nm) with high image quality.
More desirably, if the reflectance is 98% or more in a region of 90% or more of these reflection wavelength bands, the wavelength characteristics can be regarded as sufficiently uniform, and the wavelength depends on the incident angles of the dichroic mirrors 10D and 13D. Change in characteristics (reflectance) is small. Thereby, the fluorescence image data of the observation area | region of the sample 16 is acquirable with a favorable SN ratio in the whole visual field.

  The characteristics of the dichroic mirrors 9, 10D, 13D, the first filter 10E, and the second filter 10F are desirably set according to the combination of light sources to be mounted. Incidentally, when the number of combinations of light sources is large, a dichroic mirror with various characteristics and a plurality of types of filters may be prepared and used properly. A plurality of types of dichroic mirrors and a plurality of types of filters may be attached to each of the optical path switching units 10 and 13 in advance.

  In the microscope main body 100 described above, there are three types of optical elements (setting elements) mounted on the optical path switching units 10 and 13: a dichroic mirror, a total reflection mirror, and a hollow block. It is good also as two types. Moreover, it is good also as only a dichroic mirror. Further, instead of the dichroic mirror (or in addition to the dichroic mirror), other types of beam splitters such as a half mirror and a multiband dichroic mirror (a dichroic mirror having a plurality of transmission wavelength bands and reflection wavelength bands) may be provided. . When a multi-band dichroic mirror or a half mirror is used, a plurality of combinations of a laser light source for light stimulation and a laser light source for imaging can be used in a single dichroic mirror or half mirror.

  If the optical element arranged at the branch point of the optical paths R1 and R2 is a half mirror and it is not necessary to switch to another optical element, the half mirror is arranged as it is without using the optical path switching units 10 and 13. May be.

  Further, although the above-described microscope main body 100 is equipped with different types of galvano scanners (a control type galvano scanner 11 and a resonance type galvano scanner 12) as a pair of galvano scanners, the same type of galvano scanner is mounted. Also good. However, it is more preferable to install different types of galvano scanners because it gives freedom to use them differently (and is not limited to galvano scanners, and members that can change the direction of light, such as acousto-optic elements, may be used. .)

  Further, in the microscope main body 100 described above, the number of optical path separations (the number of switching) and the number of galvano scanners are each 2, but it may be expanded to 3 or more to further increase the degree of freedom of observation.

  The microscope main body 100 described above is a laser scanning microscope equipped with both a fluorescence detection function and a confocal detection function, but does not include one or both of the fluorescence detection function and the confocal detection function. The present invention can also be applied to a laser scanning microscope.

[Fourth Embodiment]
Finally, as a fourth embodiment, an example of a light stimulus observation method using a half mirror will be briefly described. Here, description will be made on the assumption that the setting content of the microscope main body 100 is the same as that of the third embodiment.

  The procedure of light stimulus observation is as shown in FIG.

(Step 1)
The controller 20 sets the use light source of the laser unit 1 to the laser light source 3 (argon laser light source) and sets the setting elements of the optical path switching units 10 and 13 to half mirrors. At this time, the intensity of the emitted light from the laser unit 1 is set to an imaging intensity (low intensity).

    Further, the controller 20 removes at least one galvano mirror of the control type galvano scanner 11 from the optical path.

(Step 2)
The controller 20 drives the laser unit 1, the resonant galvano scanner 12, and the photodetector 19, and performs imaging of the entire observation region of the specimen 16 with low-intensity argon laser light. The fluorescence image data of the specimen 16 acquired by imaging is sent to the computer 21.

(Step 3)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22 and causes the user to designate one or a plurality of partial areas on which light stimulation should be performed on the screen. The user operates the input device 23 and designates a desired partial area to the computer 21. Information on the partial area designated by the user is sent to the controller 20.

(Step 4)
The controller 20 sets the scan area of the control type galvano scanner 11 to one or a plurality of partial areas designated by the user. Moreover, the controller 20 changes the intensity | strength of the emitted light of the laser unit 1 to the intensity | strength for light stimulation (high intensity | strength).

  The controller 20 also removes at least one galvanometer mirror of the resonance type galvano scanner 12 from the optical path.

(Step 5)
The controller 20 drives the laser unit 1 and the control type galvano scanner 11 and applies light stimulation to a partial region of the specimen 16 with high-intensity argon laser light. At this time, it is not necessary to drive the photodetector 19.

(Step 6)
The controller 20 changes the intensity of the laser light source 3 (argon laser light source) of the laser unit 1 to low intensity (for imaging).

    Further, the controller 20 removes at least one galvano mirror of the control type galvano scanner 11 from the optical path.

(Step 7)
The controller 20 drives the laser unit 1, the resonant galvano scanner 12, and the photodetector 19, and performs imaging of the entire observation region of the specimen 16 with low-intensity argon laser light. The acquired fluorescence image data of the specimen 16 is sent to the computer 21.

(Step 8)
The computer 21 outputs the fluorescence image data of the specimen 16 to the monitor 22. On the screen, the user can observe the state of the specimen 16 immediately after the light stimulus. The computer 21 stores the fluorescence image data as necessary (step 8).

  Each step described above is repeated or returned to the previous step as necessary.

  In step 5 described above, the light stimulation is performed only once, but may be performed repeatedly in succession. Then you can stimulate more strongly.

Note that in step 7 described above, imaging was performed only once, but may be performed repeatedly in succession. By doing so, fluorescent image data for a plurality of consecutive frames can be obtained, and thus it is possible to observe the temporal change of the specimen 16 immediately after the light stimulation.

It is a block diagram of the system of 1st Embodiment. It is a figure which shows the procedure of 2nd Embodiment. It is a conceptual diagram explaining the optical path of the argon laser beam in 2nd Embodiment. It is a conceptual diagram explaining the optical path of the ultraviolet laser beam in 2nd Embodiment. It is a figure which shows the procedure of 3rd Embodiment. It is a conceptual diagram explaining the optical path of the low intensity | strength argon laser beam in 3rd Embodiment. It is a conceptual diagram explaining the optical path of the high intensity | strength argon laser beam in 3rd Embodiment. It is a figure which shows the procedure of 4th Embodiment. (A) It is a figure explaining the structure of an optical path switching unit, (b) is a figure explaining the structure of the optical path switching unit which shows the arrow A of (a).

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Laser unit, 7 ... Optical fiber, 8 ... Collimating lens, 9 ... Dichroic mirror, 10, 13 ... Optical path switching unit, 11 ... Control type galvano scanner, 50, 51 ... Turret, 52 ... Small gear, 53 ... Motor 61 62 ... Filter holding member

Claims (11)

An optical path dividing means for guiding light from the light source section to the surface to be observed and guiding light from the surface to be observed to the detector;
An optical path that is disposed between the optical path dividing means and the surface to be observed and that separates an optical path between the optical path dividing means and the surface to be observed between a first optical path and a second optical path having different paths. Switching means;
A first scanner disposed in the first optical path;
A second scanner disposed in the second optical path and driven independently of the first scanner,
The optical path switching means is
A first optical path switching unit disposed at one branch point of the first optical path and the second optical path;
A second optical path switching unit disposed at the other branch point of the first optical path and the second optical path,
And a plurality of filters disposed between the first optical path switching unit and the first scanner and between the first optical path switching unit and the second scanner, respectively. Laser scanning microscope. The filter is a laser scanning microscope according to claim 1, characterized in that it is held by a holding member provided on said first optical path switching unit. It said first optical path switching unit and the second optical path switching unit, a mirror, a laser scanning microscope according to claim 1 or 2, characterized in that it comprises at least one beam splitter. 4. The laser scanning microscope according to claim 3, wherein the beam splitter is a dichroic mirror having a plurality of transmission bands. Said beam splitter, a reflection band, a dichroic mirror having a transmission band on the short wavelength side of the reflection band, the reflection band is to claim 4, characterized in that the reflectance of 95% or more at wavelength width 70nm or more The laser scanning microscope described. Said beam splitter, a reflection band, a dichroic mirror having a transmission band on the short wavelength side of the reflection band, the reflection band is to claim 4, characterized in that the reflectance of 95% or more than the wavelength width 130nm The laser scanning microscope described. Said beam splitter, a reflection band, a dichroic mirror having a transmission band on the short wavelength side of the reflection band, the reflection band is to claim 4, characterized in that the reflectance of 95% or more in the wavelength range 260nm or more The laser scanning microscope described. The laser scanning microscope according to any one of claims 1 to 7 , wherein the dichroic mirror has a reflectance of 98% or more in a region of 90% or more of a reflection wavelength band. The first optical path switching unit and the second optical path switching unit are formed of turrets having gears having the same number of teeth, and are adjacently arranged so that the gears mesh with each other . 9. A laser scanning microscope according to 8 . The light source unit is
Different types of light sources wavelengths, according to any one of claim 1 to 9, characterized in that it comprises one light source for emitting a plurality of infrared light sources, or a plurality of infrared light of different wavelengths Laser scanning microscope. The first scanner and the second scanner at least one laser scanning microscope according to any one of claim 1 to 10, characterized in that a resonant galvanometer scanner.

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