JPH10197799A - Optical axis deviation correcting device and scan type optical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to correct optical axis deviation at whatever angel to the optical axis an excited light quantity balancer is arranged and to obtain a fluorescent image of multiple dyeing which has no unevenness and is easy to observe. SOLUTION: This optical axis deviation correcting device corrects optical axis deviation caused when an excited light quantity ratio is adjusted by rotating the excited light quantity balancer 14 which is provided rotatably in the lighting optical path of an optical microscope and varies in transmission wavelength band with the angle of rotation. An optical element 15 for optical axis correction is arranged rotatably in the projection-side focal optical system of the excited light quantity balancer 14 and then rotated associatively with the rotation of the excited light quantity balancer 14 so as to correct the optical axis deviation caused by the rotation of the excited light quantity balancer 14.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning optical microscope provided with an excitation light amount balancer for adjusting the light intensity ratio of multi-wavelength excitation light and an optical axis shift caused by adjusting the light intensity ratio of illumination light of the optical microscope. The present invention relates to an optical axis shift correction device that corrects the optical axis deviation.

[0002]

2. Description of the Related Art In recent years, in the fields of medicine, biology, and the like, fluorescent specimens stained with a plurality of fluorescent dyes have been frequently observed. Techniques have been developed to efficiently excite multiple-stained fluorescent specimens and to arbitrarily change the brightness ratio of each fluorescent image corresponding to the fluorescent dye.

FIG. 11 shows an outline of an epi-illumination fluorescence microscope described in Japanese Patent Application Laid-Open No. 5-150164.
The epi-illumination fluorescence microscope converts the light from the epi-illumination light source 60 into a plurality of narrow-band excitation light wavelengths through an excitation filter 61, and converts the light having the plurality of narrow-band excitation light wavelengths into an objective lens 62.
Then, the sample 53 is irradiated. A plurality of types of fluorescence emitted from the specimen 63 are transmitted through the dichroic mirror 64 and enter the absorption filter 65. A transmission characteristic is set in the absorption filter 65 so as not to transmit an extra wavelength range than light transmitted by the dichroic mirror 64 and to transmit only the wavelength of the fluorescent image. Therefore, the absorption filter 6
The fluorescence image transmitted through 5 is guided to an eyepiece observation optical system or a photodetector (not shown) so that the fluorescence image can be observed.

In the above-mentioned epi-illumination fluorescence microscope, a transmission wavelength shifting interference filter 66 for changing the light amount ratio of a plurality of narrow band excitation lights is arranged in the excitation light path before the dichroic mirror 64. As shown in FIGS. 12A and 12B, the transmission wavelength band of the transmission wavelength shifting interference filter 66 shifts according to the inclination. The light amount ratio can be changed.

According to the above-described epi-illumination fluorescence microscope, by rotating the interference filter 66 so as to be inclined gradually, the excitation light amount ratio of each fluorescent dye emitted from the multi-stained fluorescent specimen 63 can be easily adjusted, and the multi-stained fluorescent The brightness ratio of the image can be changed arbitrarily.

[0006]

However, if the inclination of the interference filter with respect to the optical axis is adjusted to adjust the excitation light amount ratio of the fluorescent dye, the thickness of the interference filter causes the thickness of the interference filter to be adjusted with respect to the optical axis. When the light is inclined, the transmitted light is refracted and causes an optical axis shift with respect to the illumination light. In general, an epi-illumination microscope is designed so that an optical system can project an arc of an illumination light source to the center of an objective pupil in order to obtain a uniform image.
However, in the above fluorescence microscope, since the optical axis shifts due to the tilting of the interference filter, the arc of the illumination light source is projected with a shift with respect to the objective pupil. As a result, when the excitation light amount ratio is adjusted, the fluorescent image becomes uneven.

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides an optical axis deviation correcting device capable of correcting an optical axis deviation of light transmitted through an excitation light amount balancer. It is an object of the present invention to provide a scanning optical microscope provided with an optical axis deviation correcting device.

[0008]

In order to achieve the above object, the present invention takes the following measures. The present invention is directed to a light for correcting an optical axis shift generated when an excitation light amount balancer is adjusted by rotating an excitation light amount balancer rotatably provided in an illumination optical path of an optical microscope and having a transmission wavelength band that changes according to a rotation angle. An axis deviation correcting device, wherein an optical element for optical axis correction is rotatably arranged in a focal optical system on an emission side of the excitation light amount balancer, and an optical axis deviation caused by rotation of the excitation light amount balancer is corrected. The optical element is rotated in conjunction with the rotation of the excitation light amount balancer.

According to the present invention, by using an optical axis deviation correcting device comprising an excitation light amount balancer and an optical element interlocked with the excitation light amount balancer in an optical microscope, the optical axis deviation of the light transmitted through the excitation light amount balancer is obtained. Can be corrected by the optical element. Therefore, even if the excitation light amount balancer is arranged at any angle with respect to the optical axis, the deviation of the optical axis is corrected, so that the ratio of the brightness of the fluorescent image can be arbitrarily changed, and it is easy to observe. Fluorescent images with no staining can be obtained.

Also, a machine for mechanically connecting an excitation light amount balancer and an optical axis correcting optical element and rotating the optical axis correcting optical element by an optical axis shift caused by rotation of the excitation light amount balancer. It has a typical interlocking mechanism.

Further, the present invention converts illumination light having a plurality of wavelengths into a plurality of excitation wavelengths through an excitation filter, and changes a transmission wavelength band by rotating an excitation light amount balancer disposed on the incident side of the excitation filter. In a scanning optical microscope in which the excitation light amount ratio is adjusted and the excitation light amount ratio is adjusted and the sample is irradiated with the multi-wavelength excitation light through the scanning optical system, the light is introduced into the focal optical system on the emission side of the excitation light amount balancer. An optical element for axis correction is rotatably arranged,
The optical element is rotated in conjunction with the rotation of the excitation light amount balancer so as to correct the optical axis shift caused by the rotation of the excitation light amount balancer.

[0012]

Embodiments of the present invention will be described below. (First Embodiment) FIG. 1 shows the entire configuration of an embodiment in which the present invention is applied to an epi-fluorescence microscope.

Illumination light emitted from a light source 11 such as a mercury lamp is focused by a collector lens 12 and
, And enters the excitation light amount balancer 14. The excitation light amount balancer 14 has the same transmission characteristics as the above-described interference filter 66, and is arranged rotatably with respect to the optical axis of the illumination light. A parallel plane plate 15 that rotates in conjunction with the excitation light amount balancer 14 is arranged on the emission side of the excitation light amount balancer 14, and a field stop 16 and a projection lens 17 are arranged on the emission side of the parallel plane plate 15. The illumination light emitted from the projection lens 17 is passed through an excitation filter 18 of a fluorescent cube to convert the illumination light into a plurality of narrow-band excitation light wavelengths, reflected by a dichroic mirror 19 of the fluorescent cube to the sample S side, and reflected by an objective lens 20. Irradiates the specimen S through

On the other hand, a beam splitter 22 is disposed on the exit side of the dichroic mirror 19 viewed from the sample S side, for removing an extra wavelength and switching the optical path to an observation optical system or a photographic optical system as necessary. Further, an eyepiece optical system 23 is arranged on the observation optical path side of the beam splitter 22, while a photographing optical element 24 is arranged on the photographing optical path side.

FIG. 2 shows an optical system from the light source 11 to the objective lens 20 of the epi-fluorescence microscope, and FIG. 3 shows the interlocking relationship between the excitation light amount balancer 14 and the plane-parallel plate 15. .

The excitation light amount balancer 14 has a rotation axis perpendicular to the optical axis and perpendicular to the plane of the paper, is held by a frame 14 'having a circular outer periphery with gears, and is rotatable in the optical axis. ing. The excitation light amount balancer 14 converts light from the light source 11 into a plurality of narrow-band excitation light wavelengths, and further functions to vary the light amount ratio of the plurality of narrow-band excitation lights by rotation in the optical axis. I have. The plane-parallel plate 15 has a rotation axis perpendicular to the optical axis and perpendicular to the plane of the paper, and is held by a frame 15 'having a gear formed on a circular outer periphery. Plane plate 1 parallel to frame 14 'of excitation light amount balancer 14
5 is arranged so as to mesh with each other, so that the parallel plane plate 15 can rotate in the optical axis in conjunction with the rotation of the excitation light amount balancer 14.

Further, the arc of the light source 11 is projected onto the center of the pupil 20b of the objective lens 20 by the projection lens 17 arranged on the exit side of the plane-parallel plate 15, and illuminates the sample S through the lens element 20a. .

In the case of fluorescence observation of a multi-stained specimen, when the excitation light amount balancer 14 is rotated to adjust the excitation light amount ratio, as shown in FIG.
Is inclined in a direction parallel to the optical axis. for that reason,
The light transmitted through the excitation light amount balancer 14 is shifted from the optical axis due to refraction.

On the other hand, the parallel plane plate 15 held by the frame 15 'is engaged with the frame 14' when the frame 14 'of the excitation light amount balancer 14 is rotated to adjust the excitation light amount ratio. The frame 15 ′ of the plane-parallel plate 15 is rotated by receiving a rotational force in a direction opposite to that of the frame 14 ′ of the excitation light amount balancer 14. For example, if the excitation light amount balancer 14 is rotated clockwise, the plane parallel plate 15 is rotated counterclockwise.

Accordingly, parameters such as the refractive index and thickness of the parallel plane plate 15 and the rotation ratio between the plane parallel plate 15 and the excitation light amount balancer 14 are set in advance so that the optical axis deviation caused by the excitation light amount balancer 14 can be corrected. In other words, the optical axis shift caused by refraction when transmitting through the excitation light amount balancer 14 can be returned to the original optical axis by the refraction received when transmitting through the parallel plane plate 15. The deviation of the optical axis can be corrected by the plane parallel plate 15.

As described above, according to the present embodiment, when the excitation light amount balancer 14 is rotated to adjust the excitation light amount ratio or the like, the excitation light amount balancer 14 is disposed at any angle with respect to the optical axis. The light from the light source 11 can be guided to the objective lens 20 without any misalignment, and the arc of the light source 11 can always be projected on the center of the pupil 20b. As a result, efficient excitation can be performed, the brightness ratio of the fluorescent image can be arbitrarily adjusted, and a multi-stained fluorescent image can be obtained without unevenness and easy to observe.

(Second Embodiment) Excitation Light Balancer 1
The rotation of the plane parallel plate 15 is interlocked with the rotation operation of the excitation light amount balancer 14 by disposing the plane 4 and the plane parallel plate 15 separately and electrically connecting them.

FIG. 4 shows a partial configuration of the second embodiment. The epi-fluorescence microscope according to the present embodiment has the arrangement position of the excitation light amount balancer 14 and the excitation light amount balancer 1.
The configuration is the same as that of the first embodiment, except for an interlocking mechanism between 4 and the parallel flat plate 15.

An excitation light amount balancer 14 is rotatably provided on the optical path in front of the aperture stop 13.
4 is detected by the encoder 25. A parallel flat plate 15 is rotatably provided on the optical path on the emission side of the aperture stop 13, and the rotation of the parallel flat plate 15 is rotationally driven by a motor 26. The detection signal output from the encoder 25 is input to the control unit 27, and the rotation direction and the rotation angle of the excitation light amount balancer 14 are detected. Then, the control unit 27 sets the rotation direction and rotation angle of the excitation light amount balancer 14 to the optical axis deviation caused by the rotation of the excitation light amount balancer 14 and the parallel plane plate 15.
Is converted into a rotation direction and a rotation angle of the parallel flat plate 15 which can be corrected by the above. The rotation of the plane parallel plate 15 is controlled by controlling the motor 26 based on the rotation direction and the rotation angle of the plane parallel plate 15 thus determined.

As described above, the excitation light amount balancer 14 and the parallel plane plate 15 are electrically connected to each other to
By interlocking the rotation of the parallel plane plate 15 with the rotation of the optical axis, the optical axis deviation caused by the rotation of the excitation light amount balancer 14 can be corrected, and the parallel plane plate 15 is arranged at a position away from the excitation light amount balancer 14. Because it is possible, the degree of freedom of design increases.

(Third Embodiment) In a third embodiment, the present invention is applied to an epi-illumination fluorescence microscope similarly to the first embodiment, and a wedge-shaped prism is used instead of a plane-parallel plate. You are using

FIG. 5 shows the configuration of a portion from the light source to the objective lens of the epi-illumination fluorescence microscope according to the third embodiment. The same or corresponding parts as those in the first embodiment are denoted by the same reference numerals.

A wedge-shaped prism 31 is arranged adjacent to the excitation light amount balancer 14. A wedge-shaped prism 3 is attached to an L-shaped frame 31 'held by a guide (not shown) so as to be movable in a direction parallel to the optical path before the prism in the direction of the arrow shown in the figure.
Holds 1. A frame 14 'for holding the excitation light amount balancer 14 is provided on a rack formed on the L-shaped frame 31'.
The excitation light amount balancer 14 (frame body 14 ') and the L-shaped frame 31' are interlocked by meshing the gears formed on the outer periphery of.

Since the output light of the wedge-shaped prism 31 is always inclined at a certain angle with respect to the incident light, the optical axis of the optical path before the prism 31 is inclined at a certain angle after the prism 31. Then, the light from the light source 11 is guided to the objective lens 20 and the arc of the light source 11 is
The prism 31 is projected onto the center of the pupil 20b
The optical system is designed to tilt the optical axis of the previous optical path.

If the state in which the excitation light amount balancer 14 is perpendicular to the optical axis is set as the reference state, the light transmitted through the excitation light amount balancer 14 in the reference state coincides with the optical axis emitted from the wedge prism 31. The wedge-shaped prism 31 is moved in parallel with the optical axis before the prism in conjunction with the rotation of the excitation light amount balancer 14. In other words, as shown by the solid line in FIG. 6, the wedge-shaped prism 31 is coupled with the rotation of the excitation light amount balancer 14 until the emission position coincides with the optical axis of the light emitted from the wedge-shaped prism 31 in the reference state. Translate parallel to previous optical axis.

As described above, if the moving distance of the wedge prism 31 is set in conjunction with the rotation of the excitation light amount balancer 14, the optical axis shift caused by the rotation of the excitation light amount balancer 14 in the optical axis can be reduced. 31 can be corrected by the parallel movement.

According to such an embodiment, the same effects as those of the first embodiment can be obtained, and the wedge-shaped prism 31 is moved in parallel, so that the interlocking mechanism can be configured in a small space. .

(Fourth Embodiment) Excitation Light Balancer 1
4 and the wedge-shaped prism 31 are not mechanically connected by using a rack and pinion mechanism, but are electrically connected and linked in the same manner as in the second embodiment.

FIG. 7 shows a configuration from the light source 11 to the objective lens 20 of the epi-illumination fluorescence microscope according to the fourth embodiment. This embodiment is configured in the same manner as the third embodiment except for the arrangement position of the wedge prism 31 and the interlocking mechanism between the excitation light amount balancer 14 and the wedge prism 31.

An excitation light amount balancer 14 is rotatably provided on the optical path in front of the aperture stop 13.
4 is detected by the encoder 25. Further, a holding member 31a holding the wedge-shaped prism 31 on the optical path on the emission side of the aperture stop 13 is held by a guide (not shown) so as to be movable in a direction parallel to the optical axis of the path before the prism. The movement is performed by the driving force of the motor 26.

The detection signal output from the encoder 25 is input to the control unit 27 to detect the rotation direction and rotation angle of the excitation light amount balancer 14. Then, the control unit 27 converts the rotation direction and the rotation angle of the excitation light amount balancer 14 into a movement amount capable of correcting the optical axis shift caused by the rotation of the excitation light amount balancer 14 by the wedge prism 31. By controlling the motor 26 based on the amount of movement of the wedge prism 31 determined in this way, the wedge prism 31
Is translated.

As described above, the excitation light amount balancer 14 and the wedge prism 31 are electrically connected to each other, and the rotation of the excitation light amount balancer 14 is linked to the rotation of the excitation light amount balancer 14, whereby the rotation of the excitation light amount balancer 14 is performed. Can be corrected, and the wedge-shaped prism 31 can be arranged at a position distant from the excitation light amount balancer 14, so that the degree of freedom in design increases.

(Fifth Embodiment) The fifth embodiment is applied to an epi-fluorescence microscope similarly to the first embodiment, and is different from the first embodiment in that a parallel plane plate is used. Then, a projection lens is used as an optical element for optical axis correction.

FIG. 8 shows the configuration from the light source 11 to the objective lens 20 in the epi-illumination fluorescence microscope according to the present embodiment, and the portions having the same functions as those in the first embodiment are denoted by the same reference numerals. .

The arc of the light source 11 is connected to the pupil 2 of the objective lens 20.
The projection lens 17 for projecting the optical axis 0b functions as an optical element that corrects the optical axis shift. A frame 17 'holding the projection lens 17 is movably held by a guide (not shown) in a direction orthogonal to the optical axis. A long hole 42 is formed in one end of the connecting rod 41 for interlocking the projection lens 17 and the excitation light amount balancer 14 in the longitudinal direction, and one end of the connecting rod 41 is projected by a pin 43 slidable in the long hole 42. The lens 17 is connected to the upper part of the frame 17 '. Further, a similar long hole 44 is formed at the other end of the connecting rod 41, and the other end of the connecting rod 41 is extended at the other end of the connecting rod 41 in a direction orthogonal to the optical axis by a pin 45 slidable in the long hole 44. Connected to the department. The gear of the frame 14 ′ of the excitation light amount balancer 14 is meshed with the rack 46. By pivotally supporting the intermediate portion of the connecting rod 41 with the pin 47, the connecting rod 41 can rotate about the pin 47.

In the above-described configuration, the rotation of the excitation light amount balancer 14 causes a shift in the optical axis as shown in FIG. 9, and the rack 46 is moved in conjunction with the rotation of the frame 14 'of the excitation light amount balancer 14. Move up and down. One end of the tie rod 41 connected to the rack 46 at one end is lifted or lowered at one end in conjunction with the vertical movement of the rack 46. As a result, the end of the connecting rod 41 on the projection lens side moves in the opposite direction to the rack 46 with the intermediate pin 47 as a fulcrum. The frame 17 'of the projection lens 17 connected to the projection lens side end of the tie rod 41 by a pin 43 is lifted or lowered in a direction perpendicular to the optical axis. As shown in FIG. 9, for example, when the excitation light amount balancer 14 rotates in a direction to shift the optical axis downward, the projection lens 17 moves downward, and the light emitted from the projection lens 17 has the optical axis deviation corrected. State.

According to such an embodiment, the optical axis shift caused by the rotation of the excitation balancer 14 in the optical axis can be corrected by the parallel movement of the projection lens 17, and the number of optical components used is small. Therefore, there is also an effect that loss of light amount can be reduced.

(Sixth Embodiment) FIG. 10 shows a partial configuration of an embodiment in which the present invention is applied to a scanning microscope. Reference numeral 51 denotes a multi-wavelength laser beam whose beam width is adjusted by a beam expander 52. Reference numeral 53 denotes an excitation dichroic mirror, which converts a laser beam into an excitation wavelength and reflects the laser light toward the sample. A scanning optical system is arranged on the optical path from the excitation dichroic mirror 53 to the objective lens. The scanning optical system is arranged at a position conjugate with the pupil of the objective lens, and includes an X galvano 54 and a Y galvano 55 for scanning the sample with laser light. The fluorescence emitted from the sample and transmitted through the excitation dichroic mirror 53 is imaged by the imaging lens 56 at the position of the pinhole 57. Then, the fluorescence passing through the pinhole 57 is detected by the photomultiplier 58.

The excitation light amount balancer 14 and the plane-parallel plate 15 having the same mechanism as that described in the first embodiment are arranged on the optical path between the excitation dichroic mirror 53 and the beam expander 52. .

In the scanning microscope configured as described above, the laser beam 51 passes through the beam expander, is reflected by the excitation dichroic 52, and
And proceed to Y galvano 55. Further, the light is irradiated onto the specimen through an objective lens (not shown). The sample is scanned by driving the X galvo 54 and the Y galvo 55 in synchronization.

The excited fluorescence of the sample passes through the objective lens, passes through the X galvo 54 and the Y galvo 55,
The light is guided to the excitation dichroic mirror 53. After passing through the excitation dichroic mirror 53, an image is formed at the position of the pinhole 57 by the imaging lens 56. The light passing through the pinhole 54 is detected by the photomultiplier 58, and a scanned image is formed from the output signal of the photomultiplier 58.

Here, in order to form a scanning microscope image without unevenness, it is necessary that the laser beam is always guided to the center of the galvano and the center of the pupil of the objective lens at the conjugate position of the galvano. In particular, when adjusting the light amount ratio of the multi-wavelength excitation light using the excitation light amount balancer 14, the above-described condition may not be satisfied because the above-described optical axis shift occurs due to the rotation of the excitation light amount balancer 14.

Therefore, as described in the first embodiment, since the optical axis shift caused by the rotation of the excitation light amount balancer 14 is corrected by the parallel flat plate 15 interlocking with the excitation light amount balancer 14, the multi-stained fluorescent specimen When the scanning fluorescent image is formed, even if the excitation light amount balancer 14 is rotated to shift the wavelength of the multi-wavelength laser light and the excitation light amount is arbitrarily adjusted, there is no optical axis shift, and the laser light is always supplied to the galvanometer and the objective lens. Can be led to the center of the pupil, and as a result, a multi-stained fluorescent image that is easy to observe without unevenness can be obtained. The present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

[0049]

As described above in detail, according to the present invention, by correcting an optical axis shift by an optical element interlocking with the excitation light amount balancer, the angle of the excitation light amount balancer with respect to the optical axis can be increased. Can correct the optical axis shift, arbitrarily change the brightness ratio, and obtain a multi-stained fluorescent image which is easy to observe without unevenness.

[Brief description of the drawings]

FIG. 1 is an overall configuration diagram of an epi-fluorescence microscope according to a first embodiment.

FIG. 2 is a diagram illustrating a configuration from a light source to an objective lens according to the first embodiment.

FIG. 3 is a principle diagram for correcting an optical axis shift in the first embodiment.

FIG. 4 is a configuration diagram from a light source to an objective lens of an epi-illumination fluorescence microscope according to a second embodiment.

FIG. 5 is a configuration diagram from a light source to an objective lens of an epi-illumination fluorescence microscope according to a third embodiment.

FIG. 6 is a principle diagram for correcting an optical axis shift according to the third embodiment.

FIG. 7 is a configuration diagram from a light source to an objective lens of an epi-illumination fluorescence microscope according to a fourth embodiment.

FIG. 8 is a configuration diagram from a light source to an objective lens of an epi-illumination fluorescence microscope according to a fifth embodiment.

FIG. 9 is a principle diagram for correcting an optical axis shift in a fifth embodiment.

FIG. 10 is a partial configuration diagram of a scanning microscope according to a sixth embodiment.

FIG. 11 is a configuration diagram of a conventional epi-illumination fluorescence microscope.

FIG. 12 is a filter characteristic diagram of an excitation filter and an excitation light amount balancer.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 ... Light source 12 ... Collector lens 13 ... Aperture stop 14 ... Excitation light amount balancer 15 ... Parallel plane plate 16 ... Field stop 17 ... Projection lens 18 ... Excitation filter 19 ... Dichroic mirror 20 ... Objective lens 25 ... Encoder 26 ... Motor 27 ... Control Department

Claims (3)

[Claims] An optical axis shift generated when an excitation light amount balancer is rotated by rotating an excitation light amount balancer which is rotatably provided in an illumination optical path of an optical microscope and whose transmission wavelength band changes according to a rotation angle to adjust an excitation light amount ratio. An optical axis deviation correction device, wherein an optical element for optical axis correction is rotatably arranged in a focal optical system on the emission side of the excitation light amount balancer, and an optical axis deviation caused by rotation of the excitation light amount balancer is corrected. An optical axis shift correcting device, wherein the optical element is rotated in conjunction with the rotation of the excitation light amount balancer. 2. The optical axis deviation correcting device according to claim 1, wherein the excitation light amount balancer and the optical element for optical axis correction are mechanically connected, and an optical axis deviation caused by rotation of the excitation light amount balancer. An optical axis misalignment correction apparatus, comprising: a mechanical interlocking mechanism for rotating the optical element for optical axis correction by a distance. 3. An illumination light having a plurality of wavelengths is converted into a plurality of excitation wavelengths through an excitation filter, and a transmission wavelength band is changed by rotating an excitation light amount balancer disposed on the incident side of the excitation filter, thereby changing an excitation light amount. Adjusting the ratio, in the scanning optical microscope to irradiate the sample through the scanning optical system multi-wavelength excitation light adjusted this excitation light amount ratio,
An optical element for optical axis correction is rotatably arranged in a focal optical system on the emission side of the excitation light amount balancer, and the optical element is adjusted to the excitation light amount so as to correct an optical axis shift caused by rotation of the excitation light amount balancer. A scanning optical microscope characterized in that it rotates in conjunction with the rotation of a balancer.