JP2006003747A - Optical scanning type observation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a clear image by allowing arbitrary scans over a position for imparting optical stimulus to a specimen, and by preventing a stimulating beam from being detected. <P>SOLUTION: The optical scanning type observing apparatus 1 is equipped with: a first light source 6, a first optical scanner 7 for scanning a light beam from the first light source 6; an objective optical system 5 for forming an image on a specimen A with the light beam scanned by the first optical scanner 7; a photodetector 13 for detecting return light that returns through the objective optical system 5 and the first optical scanner 7; a second light source 18; a second optical scanner 19 for scanning a light beam from the second light source 18; and an optical axis coupling means 4, for making the optical axis of the beam scanned by the second optical scanner 19, coincides with the optical axis from the first light source 6, wherein a barrier filter 10 that shields light from the first and second light sources 6, 18 and that transmits the return light from the specimen A is set in the front of the photodetector 13. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an optical scanning observation apparatus.

  Conventionally, an optical fiber microscope disclosed in Patent Document 1 has been proposed as an optical microscope for magnifying and observing a specimen such as a biological sample in a living state (in vivo). This microscope includes a first objective lens that forms an image of laser light emitted from a laser light source on one end face of an optical fiber bundle, and a second objective lens disposed on the other end face of the optical fiber bundle, and a laser. In this structure, a laser beam from a laser light source is scanned two-dimensionally on a specimen by an optical scanner.

Conventionally, there is an optical marking technique using a substance that functions by light stimulation and emits fluorescence or activates another fluorescent substance, such as a fluorescent protein or a caged compound. In this type of optical marking technology, as a microscope observation apparatus provided with a light stimulation apparatus for irradiating a specimen with stimulation light, for example, the one shown in Non-Patent Document 1 is known.
JP 2003-344777 A (FIG. 1 etc.) Atsushi Miyawaki and one other, "Special Review Optical technology using novel fluorescent protein kaede", Cell Engineering, vol.22, No.3, 2003, p316-326

However, in the microscope observation apparatus shown in Non-Patent Document 1, it is not possible to observe a fluorescent protein or the like while giving a light stimulus.
In addition, if fluorescence observation is performed while performing light stimulation, there is a problem that the stimulation light is reflected and detected at the interface of various optical components. In fluorescence observation, the fluorescence emitted from the specimen is extremely weak, but the intensity of the stimulation light used for light stimulation is relatively high, so if the stimulation light is detected, noise in the resulting fluorescence image Is disadvantageous in that a clear fluorescent image cannot be obtained.

  The present invention has been made in view of the above-described circumstances, can arbitrarily scan a position for applying a light stimulus to a specimen, and prevents the stimulation light from being detected. An object of the present invention is to provide an optical scanning observation apparatus capable of obtaining a clear image.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a first light source, a first light scanning unit that scans light from the first light source, and objective optics that forms an image of light scanned by the first light scanning unit on a sample. System, photodetector for detecting return light returning through the objective optical system and the first optical scanning unit, a second light source, and a second optical scanning for scanning light from the second light source And an optical axis coupling means for causing the optical axis of the light scanned by the second optical scanning unit to coincide with the optical axis from the first light source. Provided is an optical scanning observation device provided with a barrier filter that blocks light from a light source and a second light source and transmits return light from the specimen.

  According to the present invention, the light emitted from the first light source is scanned by the first light scanning unit and imaged on the specimen by the objective optical system. The light generated by irradiating the sample with light from the first light source is detected by the photodetector as return light that returns via the objective optical system and the first optical scanning unit, while the second The light emitted from the light source is made to coincide with the optical axis from the first light source by the operation of the optical axis coupling means, and is imaged on the specimen by the objective optical system. In this case, since the light from the second light source is scanned by the second light scanning unit, the light from the first light source is scanned on the specimen independently of the search range by the first light scanning unit. It is possible to apply a light stimulus by irradiating a desired position of the light source.

  Further, a part of the light from the first light source and the light from the second light source is reflected at the interface of the optical component arranged until the specimen is irradiated and the return light and the light detector. Although it goes in the direction, since the barrier filter is provided in the front stage of the photodetector, the light from the first and second light sources is blocked and only the return light can be detected by the photodetector. It becomes possible. Therefore, it is possible to reduce noise in the obtained image and obtain a clear image.

In the above-described invention, a condensing lens that forms an intermediate image of light from the first light source is provided, and the first optical scanning unit rotates a pinhole disk disposed in the vicinity of the intermediate image position. It may be a disk type optical scanning unit.
By doing in this way, the light from the 1st light source can be rapidly scanned on a sample by many pinholes provided in the pinhole disk. As a result, it is possible to observe a change in the response of the specimen to the light stimulus by irradiating light from the second light source.

Further, in the above invention, a condensing lens for forming an intermediate image of light from the first light source is provided, and the first optical scanning unit is disposed at the intermediate image position, and the intermediate image is simultaneously displayed. A scanning mirror element formed by arranging a plurality of mirrors that can be selectively turned on and off can be provided.
In this way, by selectively turning on and off the scanning mirror element, it is possible to partially reflect light from the first light source that forms the intermediate image and irradiate the specimen with the objective optical system. The return light generated in the specimen returns through the objective optical system, and only the light reflected by the mirror in the on state of the scanning mirror element is detected by the photodetector.

  Then, by switching the mirrors that make up the scanning mirror element according to the ON state, the irradiation position of the light to the specimen can be changed, and a wide range can be observed. By making each mirror constituting the scanning mirror element sufficiently small, each mirror can function as a confocal pinhole, and an image spreading at a predetermined depth position below the surface of the specimen can be obtained. it can.

  In this case, the on / off state of the mirror of the scanning mirror element can be arbitrarily changed, and by switching the on / off state, the same effect as exchanging a plurality of confocal pinhole members can be achieved. In other words, if you want to obtain a high-resolution confocal image, reduce the number of mirrors that are turned on at a time, and if you want to obtain a bright and deep image, turn on multiple adjacent mirrors simultaneously. It can respond by doing.

  According to the scanning mirror element, the on / off state can be switched at high speed. Therefore, it is possible to observe the change in the response of the specimen to the light stimulus by irradiating the light from the second light source with high resolution.

Furthermore, in the said invention, it is preferable that a said 2nd optical scanning part is provided with the acoustooptic element which changes a diffraction angle according to the galvanometer mirror or the input vibration frequency.
In this way, the light from the second light source can be moved to an arbitrary position on the specimen, and can be continuously irradiated while being fixed at a desired position.

  According to the present invention, the light from the first light source for observation and the light from the second light source for light stimulation are prevented from being detected by the photodetector, and noise in the obtained image is reduced. The effect is that a clear image can be obtained.

Hereinafter, an optical scanning observation apparatus 1 according to an embodiment of the present invention will be described with reference to FIG.
As shown in FIG. 1, the optical scanning observation apparatus 1 according to the present embodiment includes an observation optical system 2, a light stimulation optical system 3, and a dichroic mirror 4 that couples the optical axes of these optical systems 2 and 3. (Optical axis coupling means) and a condensing optical system 5 that irradiates the specimen A with light from the observation optical system 2 and the light stimulation optical system 3.

  The observation optical system 2 includes a first laser light source 6 that generates laser light, a first light scanning unit 7 that scans the laser light emitted from the first laser light source 6, and the first light. A first pupil projection lens 8 that focuses the laser beam scanned by the scanning unit 7 to form an intermediate image, and a sample A that returns from the first pupil projection lens 8 and the optical scanning unit 7. A dichroic mirror 9 that branches the return light from the optical path, a barrier filter 10 that transmits only the fluorescence from the specimen A among the branched return light, and a confocal lens 11 that collects the fluorescence that has passed through the barrier filter 10. And a confocal pinhole 12 disposed in the vicinity of an image formation position by the confocal lens 11 and a photodetector 13 for detecting fluorescence that has passed through the confocal pinhole 12. In the figure, reference numerals 14 and 15 are dichroic mirrors, and reference numeral 16 is a bandpass filter.

The first optical scanning unit 7 is a so-called proximity galvanometer mirror composed of two galvanometer mirrors 7 a and 7 b that can swing around two orthogonal axes, and is emitted from the first laser light source 6. The laser beam is configured to scan two-dimensionally on one end surface 17a of an optical fiber bundle 17 to be described later.
The photodetector 13 is, for example, a photomultiplier tube (PMT).

  In FIG. 1, a plurality of first laser light sources 6 are provided, but this is for enabling the sample A to be irradiated with laser beams having different wavelengths, and a single laser light source 6 may be used. In addition, although an example in which a plurality of photodetectors 13 are provided and the fluorescence of different wavelengths is separately detected by changing the wavelength of the bandpass filter 15 is shown, a single photodetector 13 may be used. .

  As described above, the barrier filter 10 allows the fluorescence generated in the specimen A to pass therethrough, and in particular blocks the laser light emitted from the first laser light source 6 and the second laser light source 18 described later. It is configured.

The optical stimulation optical system 3 includes a second laser light source 18 that generates laser light, a second optical scanning unit 19 that scans the laser light emitted from the second laser light source 18, and the first And a second pupil projection lens 20 that focuses the laser light scanned by the second optical scanning unit 19 to form an intermediate image. Reference numeral 21 denotes a dichroic mirror.
The second laser light source 18 emits laser light having a wavelength different from that of the first laser light source 6. In the drawing, a plurality of second laser light sources 18 are also provided, but this is for enabling the sample A to be irradiated with laser beams having different wavelengths, and may be a single laser light source.
Similarly to the first optical scanning unit 7, the second optical scanning unit 19 is also configured by a proximity galvanometer mirror in which two galvanometer mirrors 19 a and 19 b are arranged close to each other.

The condensing optical system 5 is focused by the imaging lens 22 and the imaging lens 22 that collects the laser light that forms an intermediate image by the first and second pupil projection lenses 8 and 20. The first objective lens 23 that forms an image of the laser light, the optical fiber bundle 17 that arranges one end surface 17a at the image forming position by the first objective lens 23, and the other end surface 17b of the optical fiber bundle 17 are arranged. And a second objective lens 24.
The second objective lens 24 forms an image of the laser light propagated by the optical fiber bundle 17 on the observation target portion of the specimen A such as an experimental small animal disposed opposite to the tip of the second objective lens 24. It has become.

The operation of the optical scanning observation apparatus 1 according to the present embodiment configured as described above will be described below.
In order to observe the specimen A with the optical scanning observation apparatus 1 according to the present embodiment, the observation optical system 2 is operated, and the laser light emitted from the first laser light source 6 is transmitted through the dichroic mirrors 9 and 15. The light is passed through and incident on the first optical scanning unit 7. The first light scanning unit 7 scans the laser light two-dimensionally by changing the angles of the two galvanometer mirrors 7a and 7b. The scanned laser light is once condensed by the pupil projection lens 8 to form an intermediate image, passes through the dichroic mirror 4, and the one end surface of the optical fiber bundle 17 by the imaging lens 22 and the first objective lens 23. 17a is condensed. The laser light incident on one end surface 17 a of the optical fiber bundle 17 propagates through the optical fiber bundle 17 and is guided to the other end surface 17 b and is imaged on the sample A by the second objective lens 24.

  In this case, since the relatively large observation optical system 2 and the optical stimulation optical system 3 and the second objective lens 24 are connected by the optical fiber bundle 17, the optical fiber bundle 17 can be freely curved. The second objective lens 24 can be disposed at an appropriate position and posture with respect to the specimen A. Then, the imaging position on the specimen A is sequentially changed by the operation of the first optical scanning unit 7, and the entire observation range is scanned.

  When the sample A is irradiated with laser light, the fluorescent substance in the sample A is excited and emits fluorescence. The emitted fluorescence passes through the second objective lens 24, the optical fiber bundle 17, the first objective lens 23, the imaging lens 22, the dichroic mirror 4, the first pupil projection lens 8, and the first optical scanning unit 7. Through the same optical path, branched by the dichroic mirror 9 and directed to the photodetector 13. Since the dichroic mirror 9 allows the laser light from the first laser light source 6 to pass therethrough, most of the laser light is excluded from the fluorescence directed to the photodetector 13.

  The fluorescence directed to the photodetector 13 is collected by the confocal lens 11 and passed through the confocal pinhole 12 disposed in the vicinity of the image forming position. Since the confocal pinhole 12, the one end surface 17a of the optical fiber bundle 17 and the imaging position on the specimen A are in a conjugate relationship with each other, the fluorescence emitted from the imaging position on the specimen A is confocal. While all of 12 can be passed, the fluorescence emitted from a position slightly deviated from the imaging position cannot pass through the confocal pinhole 12. Therefore, an image of the tissue spreading in a plane including the imaging position of the laser beam on the specimen A is acquired by the photodetector 13.

  In this case, according to the present embodiment, since the dichroic mirror 9 is provided, most of the laser light is separated from the return light, but a part thereof is reflected by the dichroic mirror 9 and is detected by the photodetector. 13 However, according to the present embodiment, since the barrier filter 10 is disposed between the dichroic mirror 9 and the photodetector 13, the laser light from the first laser light source 6 is completely blocked by the barrier filter 10. Is done. Therefore, only weak fluorescence is incident on the photodetector 13, and a clear image in which noise included in the image is sufficiently reduced can be obtained.

  On the other hand, in the optical scanning observation apparatus 1 according to the present embodiment, the optical stimulation optical system 3 is operated to perform optical stimulation on the specimen A. When the photostimulation optical system 3 is operated, laser light is emitted from the second light source 18 and is condensed by the second pupil projection lens 20 via the dichroic mirror 21 and the second light scanning unit 19. The light is incident on the same optical axis as that of the laser light from the observation optical system 2 by the dichroic mirror 4. Therefore, the light from the second laser light source 18 is also irradiated onto the specimen A through the imaging lens 22, the first objective lens 23, the optical fiber bundle 17, and the second objective lens 24.

  In this case, in the optical scanning observation apparatus 1 according to the present embodiment, the second optical scanning unit 19 is configured by two galvanometer mirrors 19a and 19b, so that the galvanometer mirrors 19a and 19b swing. By adjusting the angle range, it is possible to freely adjust the range in which the light stimulus is applied to the specimen A, and to fix the galvanometer mirrors 19a and 19b at specific swing angle positions, to light the specimen A. Can be fixed at an arbitrary position. That is, the light stimulus location can be arbitrarily set regardless of the observation range by the observation optical system 2.

Then, by observing the specimen A by operating the observation optical system 2 while applying a light stimulus to the set desired position or range, the reaction of the specimen A to the light stimulus can be observed.
Further, part of the laser light from the second light source 18 also includes various optical components, that is, the dichroic mirror 4, the imaging lens 22, the first objective lens 23, and the end faces 17 a and 17 b of the optical fiber bundle 17. The light is reflected at the interface of the second objective lens 24 and the like and is directed to the photodetector 13, but is incident on the photodetector 13 by the operation of the barrier filter 10 provided in the front stage of the photodetector 13. Is prevented. Therefore, even if observation with the observation optical system 2 is performed while applying a light stimulus, laser light is prevented from being incident on the photodetector 13, so that a clear image with less noise can be obtained. is there.

  In the present embodiment, the galvanometer mirrors 19a and 19b are illustrated as the second optical scanning unit 19, but instead of this, an acousto-optic device (not shown) that changes the diffraction angle in accordance with the input vibration frequency. (Omitted) may be adopted. According to the acousto-optic device, by fixing the vibration frequency to be input, the photostimulation site can be fixed at a specific position, and the photostimulation site can be moved to the luminous flux by the galvanometer mirrors 19a and 19b. Since the acoustooptic element normally changes the diffraction angle in a one-dimensional manner, in order to scan the sample A in a two-dimensional manner, two acoustooptics arranged in a direction crossing the diffraction direction. The elements may be arranged in series.

Next, an optical scanning observation apparatus 30 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the optical scanning observation apparatus 1 according to the first embodiment described above, and the description thereof is omitted.

As shown in FIG. 2, the optical scanning observation apparatus 30 according to the present embodiment uses the first optical scanning unit 31 and the photodetector 32 in the observation optical system 2 to perform the optical scanning observation according to the first embodiment. This is different from the apparatus 1.
Unlike the optical scanning observation apparatus 1 according to the first embodiment that employs a proximity galvanometer mirror, the first optical scanning unit 31 employs a disk scanning system as shown in FIG. ing. The first optical scanning unit 7 includes a condensing disk 33 and a pinhole disk 34 connected by a drum 35, and can be rotated in one direction by a motor 36.

  The condensing disk 33 has a plurality of Fresnel lenses formed on one side of the glass substrate, and the Fresnel lenses are arranged in a state of being shifted by a predetermined distance in the radial direction. The pinhole disk 34 has a plurality of pinholes on the substrate, and the pinholes are formed in a state where they are sequentially shifted by a predetermined distance in the radial direction. A dichroic mirror 37 is disposed between the condensing disk 33 and the pinhole disk 34, and the fluorescence returning through the first objective lens 23, the dichroic mirror 4 and the pinhole disk 34 is reflected by the dichroic mirror 37. Thus, the light is detected by the photodetector 32.

  The photodetector 32 (hereinafter also referred to as a CCD camera 32) is, for example, a CCD camera capable of detecting fluorescence from a wide range on the specimen A at a time. Between the dichroic mirror 37 and the CCD camera 32, the barrier filter 10 having the same wavelength characteristic as that of the first embodiment is disposed. In the figure, reference numeral 38 denotes a condenser lens, and reference numeral 39 denotes an imaging lens.

The operation of the optical scanning observation apparatus 30 according to the present embodiment configured as described above will be described below.
According to the optical scanning observation apparatus 30 according to the present embodiment, the laser light from the first laser light source 6 is allowed to pass through the condensing disk 33, the dichroic mirror 37, and the pinhole disk 34, so that a plurality of light spots are obtained. Is formed on the sample A through the imaging lens 39, the dichroic mirror 4, the first objective lens 23, the optical fiber bundle 17 and the second objective lens 24. The

  Therefore, the specimen A is scanned with a plurality of light spots, and the fluorescence generated at the plurality of points is converted into the second objective lens 24, the optical fiber bundle 17, the first objective lens 23, the dichroic mirror 4, the imaging lens 39, The same optical path is returned via the pinhole disk 34 and is branched from the optical path by the dichroic mirror 37 and directed to the CCD camera 32. Since the barrier filter 10 is disposed in front of the CCD camera 32, only the fluorescence generated at a plurality of locations of the specimen is allowed to pass through the barrier filter 10 and is condensed by the condenser lens 38 onto the CCD camera 32 and imaged. Will be.

According to the optical scanning observation apparatus 30 according to the present embodiment, since the observation optical system 2 includes the disk scanning optical scanning unit 31, the specimen A is simultaneously scanned with a plurality of light spots, and the fluorescent image of the specimen A is obtained. Can be obtained quickly.
In this case, when the fluorescence optical image is acquired by the observation optical system 2 while the photostimulation optical system 3 is operated to apply the photostimulation to a specific portion or a specific range of the specimen A, the photostimulation is applied. The time required for obtaining a fluorescent image can be shortened. Therefore, it is possible to observe the temporal change in the response of the specimen A to the light stimulus while updating the fluorescence image at short time intervals.

  In the present embodiment, the disk scanning method in which the condensing disk 33 and the pinhole disk 34 are simultaneously rotated has been described as an example, but instead, as shown in FIG. A disk scanning method in which only the disk is rotated may be employed. In the figure, reference numeral 47 is a white light source such as a xenon lamp, reference numeral 40 is a filter changer for changing the wavelength of excitation light, reference numerals 41 and 42 are condensing lenses, reference numeral 43 is an aperture stop, reference numeral 44 is a collimating lens, reference numeral Reference numeral 45 denotes a condenser lens, and reference numeral 46 denotes a filter changer for changing the wavelength of fluorescence acquired by the CCD camera 32. Similarly, the disk scanning optical scanning unit 31 ′ can quickly acquire a fluorescent image.

Next, an optical scanning observation apparatus 50 according to a third embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, portions having the same configuration as those of the optical scanning observation apparatus 50 according to each of the embodiments described above are denoted by the same reference numerals, and description thereof is omitted.

The optical scanning observation device 50 according to this embodiment includes the optical scanning observation devices 1 and 30 according to the first and second embodiments described above in the first optical scanning unit 51 provided in the observation optical system 2. It is different.
As shown in FIG. 4, the first optical scanning unit 51 of the optical scanning observation apparatus 50 according to the present embodiment is a digital mirror device in which a plurality of minute mirrors (not shown) are two-dimensionally arranged. A scanning mirror element 52 such as (DMD) and a control device 53 for controlling the scanning mirror element 52 are configured. Each mirror constituting the scanning mirror element 52 is selectively turned on / off by an operation command from the control device 53. When each mirror is placed in the ON state, the excitation light incident from the light source 47 is reflected in the optical path toward the imaging lens 39, while the return light returning from the imaging lens 39 side is in the optical path toward the CCD camera 32. Reflected in. Further, when each mirror is turned off, the excitation light and the return light are reflected in a direction deviating from these optical paths.

The scanning mirror element 52 is disposed at an intermediate image position by the condenser lens 42. The intermediate image of the light source 47 is simultaneously received by a plurality of mirrors, and is reflected in the direction of the imaging lens 39 by the mirror that is in the on state.
In the figure, reference numeral 54 denotes a photometric filter that measures the amount of fluorescent light, and reference numeral 55 denotes an excitation filter that converts light from the white light source 47 into excitation light having a predetermined wavelength.

  According to the optical scanning observation apparatus 50 according to the present embodiment configured in this way, by switching the on / off state of each mirror constituting the scanning mirror element 52 in an arbitrary operation pattern by the operation of the control apparatus 53, Similar to the case of the proximity galvanometer mirror or the disk scanning method, the specimen A can be scanned with a single or a plurality of light spots. Further, since the scanning mirror element 52 is disposed at the intermediate image position by the condenser lens 42, each mirror constituting the scanning mirror element 52 is confocal disposed at a position conjugate with the one end surface 17a of the optical fiber bundle 17. Only the fluorescence that functions as a pinhole and is emitted from a position at a predetermined depth of the specimen A can be imaged by the CCD camera 32.

On the other hand, if a plurality of adjacent mirrors among the mirrors constituting the scanning mirror element 52 are simultaneously turned on, the specimen A can be irradiated with a relatively large light spot. In this way, the confocal effect is reduced, but a bright image can be obtained by increasing the amount of fluorescence received by the CCD camera 32. Therefore, it is effective for an application where observation with a bright fluorescent image is desired even when the resolution is slightly reduced, or an application where a fluorescent image over a wide range in the depth direction is desired.
In the present embodiment, a laser light source (not shown) may be employed in place of the light source 47 and the excitation filter 55 made of a xenon lamp.

1 is an overall configuration diagram showing an optical scanning observation apparatus according to a first embodiment of the present invention. It is a whole block diagram which shows the optical scanning type observation apparatus which concerns on the 2nd Embodiment of this invention. It is a whole block diagram which shows the modification of FIG. It is a whole block diagram which shows the optical scanning type observation apparatus which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

A Specimen 1,30,50 Optical scanning observation device 4 Dichroic mirror (optical axis coupling means)
5 Objective optical system 6 First laser light source (first light source)
7 First optical scanning unit 10 Barrier filter 13 Photo detector 18 Second laser light source (second light source)
19 Second optical scanning unit 19a, 19b Galvano mirror 31, 31 'Optical scanning unit (disk type optical scanning unit)
32 CCD camera (photodetector)
33 Condensing disc (condensing lens)
34 Pinhole disk 42 Condensing lens 47 Xenon lamp (first light source)
52 Scanning mirror element

Claims (4)

A first light source;
A first light scanning unit that scans light from the first light source;
An objective optical system that forms an image of the light scanned by the first optical scanning unit on the specimen;
A photodetector for detecting return light returning through the objective optical system and the first optical scanning unit;
A second light source;
A second light scanning unit that scans light from the second light source;
Optical axis coupling means for matching the optical axis of the light scanned by the second optical scanning unit with the optical axis from the first light source,
An optical scanning observation apparatus provided with a barrier filter that blocks light from the first light source and the second light source and transmits return light from the specimen in front of the photodetector. A condenser lens that forms an intermediate image of the light from the first light source;
The optical scanning observation apparatus according to claim 1, wherein the first optical scanning unit is a disk type optical scanning unit that rotates a pinhole disk disposed in the vicinity of the intermediate image position. A condenser lens that forms an intermediate image of the light from the first light source;
2. The light according to claim 1, wherein the first optical scanning unit includes a scanning mirror element that is disposed at the intermediate image position and includes a plurality of mirrors that simultaneously receive the intermediate image and can be selectively turned on / off. Scanning observation device.   4. The optical scanning observation apparatus according to claim 1, wherein the second optical scanning unit includes a galvano mirror or an acousto-optic element that changes a diffraction angle in accordance with an input vibration frequency. .

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