JP2013003338A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the technology of a laser microscope that independently controls an emitting position and an emitting angle on the surface of a specimen.SOLUTION: The laser microscope 1 comprises: a laser light source 16 that emits a laser beam; an objective lens 10 that emits a laser beam to a specimen 11; a condenser lens 22 that condenses the laser beams onto the pupil conjugate surface of the objective lens 10; a parallel flat plate 23 that moves the laser beam in a direction parallel to a direction orthogonal to an optical axis; scanning means 24 that scans the specimen 11 by moving the laser beam to the position where it emits the specimen 11; and a control device 26 that rotates the parallel flat plate 23, thereby controlling a shift amount by which the laser beam is moved parallel.

Description

  The present invention relates to a laser microscope, and more particularly to a laser microscope that scans a specimen with a scanning means.

  As a configuration of a laser microscope used in the biological field, an excitation unit that condenses laser light on a specimen to excite the specimen, and a laser beam having a wavelength different from the laser light from the excitation means on the pupil plane of the objective lens And a light stimulating means that irradiates laser light as parallel light onto a certain region on the specimen.

The laser microscope having such a configuration can independently control the excitation position and the light stimulation position by providing the scanning means for each of the excitation means and the light stimulation means. Thereby, it is possible to observe the reaction by imaging the specimen while applying a light stimulus to an arbitrary part on the specimen.
Such a laser microscope is disclosed in Patent Document 1, for example.

JP 2009-288321 A

  By the way, the light stimulating means of the laser microscope described above is required to have a function of irradiating laser light having an approximately uniform intensity distribution that is incident at a fixed irradiation angle at each irradiation position in the scanning range.

  In a microscope, light is deflected at a position optically conjugate with the pupil position of the objective lens (hereinafter referred to as the pupil conjugate position), so that the specimen is irradiated without changing the irradiation angle of the light irradiated on the specimen. Since the irradiation position of the light to be changed can be changed, it is desirable that the scanning unit is disposed at the pupil conjugate position of the objective lens.

  However, at present, the most general configuration of scanning means for two-dimensionally scanning a sample includes a galvanometer mirror for scanning the sample in the X direction and a galvanometer mirror for scanning the sample in the Y direction. The configuration of the scanning means of the laser microscope disclosed in Patent Document 1 is also the same.

  In the scanning means having such a configuration (hereinafter referred to as a proximity galvanometer mirror unit), it is physically impossible to simultaneously arrange both galvanometer mirrors at the pupil conjugate position of the objective lens. The mirrors are arranged relatively close to each other, and the pupil conjugate position of the objective lens is positioned approximately in the middle thereof.

Therefore, when the proximity galvanometer mirror unit is used, the deflection position of light by each galvanometer mirror and the pupil conjugate position of the objective lens do not completely coincide with each other. Therefore, if the light irradiation position is changed, the sample is irradiated. The light irradiation angle also changes. That is, the irradiation position and the irradiation angle on the specimen surface cannot be controlled independently.
In light of the above circumstances, an object of the present invention is to provide a technique of a laser microscope that independently controls an irradiation position and an irradiation angle on a specimen surface.

  According to a first aspect of the present invention, a laser light source that emits laser light, an objective lens that irradiates the sample with the laser light, and the laser light is condensed on a pupil conjugate plane that is conjugate to the pupil plane of the objective lens A condensing lens; a scanning unit that scans the sample by moving an irradiation position for irradiating the sample with the laser beam; a shift unit that moves the laser beam in a direction perpendicular to the optical axis; There is provided a laser microscope comprising: a shift control unit that drives a shift unit to control a shift amount in which the laser light moves in parallel.

  According to a second aspect of the present invention, in the laser microscope according to the first aspect, the scanning unit includes a first scanning unit that scans the sample in a first direction orthogonal to the optical axis, an optical axis, And a second scanning unit that scans the sample in a second direction that is orthogonal and orthogonal to the first direction, and the shift control unit controls the shift amount according to the irradiation position. Provide a laser microscope.

  According to a third aspect of the present invention, in the laser microscope according to the second aspect, the shift control unit is configured so that the laser light irradiates the specimen at a constant irradiation angle regardless of the irradiation position. Provided is a laser microscope for controlling the shift amount.

  According to a fourth aspect of the present invention, in the laser microscope according to the third aspect, the first scanning unit is a first mirror that rotates around a first rotation axis that is orthogonal to an optical axis, The second scanning means is a second mirror that rotates around a second rotation axis that is orthogonal to the optical axis and orthogonal to the first rotation axis, and between the first mirror and the second mirror. Further, a laser microscope in which the pupil conjugate plane is located is provided.

  According to a fifth aspect of the present invention, in the laser microscope according to the third aspect, the shift control unit includes the shift amount so that a condensing position of the laser light on the pupil plane is located on an optical axis. Provide a laser microscope to control.

  According to a sixth aspect of the present invention, in the laser microscope according to the third aspect, the shift control unit is configured so that the condensing position of the laser light on the pupil plane is located at a position deviating from the optical axis. Provided is a laser microscope for controlling the shift amount.

  According to a seventh aspect of the present invention, there is provided the laser microscope according to the third aspect, wherein the shift control means controls the shift amount according to the magnification of the objective lens.

  According to an eighth aspect of the present invention, there is provided the laser microscope according to the third aspect, wherein the shift control means controls the shift amount according to the wavelength of the laser light.

  According to a ninth aspect of the present invention, in the laser microscope according to the third aspect, the shift means is a parallel plate through which the laser light is transmitted, and the shift control means has the shift means orthogonal to the optical axis. Provided is a laser microscope that rotates around a rotating axis.

  ADVANTAGE OF THE INVENTION According to this invention, the technique of the laser microscope which controls independently the irradiation position and irradiation angle in a sample surface can be provided.

1 is a diagram illustrating a configuration of a laser microscope according to Example 1. FIG. It is a figure for demonstrating the change of the condensing position of the stimulating light in the pupil conjugate plane by the operation | movement of the galvanometer mirror contained in the laser microscope illustrated in FIG. It is a figure for demonstrating the fluctuation | variation of the condensing position of the stimulating light in the pupil plane of the objective lens contained in the laser microscope illustrated in FIG. It is a figure for demonstrating the parallel movement of the stimulation light by the parallel plate contained in the laser microscope illustrated in FIG. It is the figure illustrated about the condensing position of the stimulation light which injects into the galvanometer mirror contained in the laser microscope illustrated in FIG. 1 through the parallel plate by which rotation control was carried out. It is the figure which showed the modification of the shift means contained in the laser microscope which concerns on Example 1. FIG. It is the figure which showed the other modification of the shift means contained in the laser microscope which concerns on Example 1. FIG. 6 is a diagram illustrating a part of the configuration of a laser microscope according to Embodiment 2. FIG.

FIG. 1 is a diagram illustrating the configuration of the laser microscope according to the present embodiment. The laser microscope 1 illustrated in FIG. 1 includes an observation scanning unit 5 and a stimulation scanning unit 24 that operate independently of each other, so that the sample 11 is subjected to optical stimulation on an arbitrary part on the sample 11. It is possible to observe the reaction by imaging the so-called twin scan. In addition to the scanning means 24, the laser microscope 1 includes a parallel plate 23, which is a shift means for moving the stimulation light in a direction perpendicular to the optical axis, in addition to the scanning means 24. Thereby, the laser microscope 1 can control independently the irradiation position and irradiation angle which irradiate the stimulus light with respect to the sample 11, when the control apparatus 26 controls the parallel plate 23 and the scanning means 24. .
The configuration of the laser microscope 1 will be specifically described with reference to FIG.
The laser microscope 1 includes observation means including observation excitation means, light stimulation means, and a control device 26.

  The observation means includes a laser light source 2 that emits excitation light (laser light) that excites the sample 11, a shutter 3, a dichroic mirror 4 that reflects the excitation light and transmits fluorescence, and a position at which the excitation light is condensed. The scanning means 5 that moves above and scans the specimen 11, the pupil projection lens 6 that projects the pupil of the objective lens 10 onto the scanning means 5 in combination with the imaging lens 8, and the excitation light and fluorescence are transmitted and stimulation light is transmitted. A reflecting dichroic mirror 7, an imaging lens 8 for condensing the fluorescence to form an intermediate image, a mirror 9, an objective lens 10 for condensing the excitation light on the sample surface, and the confocal aperture 13 A confocal lens 12 for condensing light, a confocal stop 13 having a pinhole formed at a position optically conjugate with the front focal position (sample 11) of the objective lens 10, and a barrier filter 14 for blocking excitation light. It includes a photodetector 15 for detecting the fluorescence that has passed through the barrier filter 14.

  The scanning unit 5 moves the collection position of the excitation light on the sample surface in the X direction orthogonal to the optical axis and scans the sample 11 in the X direction, and the light collection position of the excitation light on the sample surface. A galvanometer mirror 5b that moves in the Y direction perpendicular to the axis and the X direction and scans the sample 11 in the Y direction. The galvanometer mirror 5a and the galvanometer mirror 5b are approximately in the middle of the pupil plane 10P of the objective lens 10. They are arranged so that an optically conjugate pupil conjugate plane is formed.

  The light stimulation means includes a laser light source 16 that emits stimulation light (laser light) that stimulates the specimen 11, a shutter 17, a mirror 18, a lens 19, and a lens 20, and changes the beam diameter of the stimulation light. An optical system, a field stop 21 with a variable aperture disposed on a surface optically conjugate with the front focal plane (sample surface) of the objective lens 10, and a pupil conjugate plane conjugate with the pupil plane 10P of the objective lens 10. The specimen 11 is scanned by moving the condensing lens 22 for condensing the stimulation light, the parallel plate 23 for moving the stimulation light in a direction perpendicular to the optical axis, and the irradiation position for irradiating the specimen 11 with the stimulation light. A pupil projection lens 25 that projects the pupil of the objective lens 10 onto the scanning unit 5 in combination with the scanning unit 24 and the imaging lens 8, a dichroic mirror 7, the imaging lens 8, the mirror 9, and the sample 11. It includes an objective lens 10 for irradiating, a. The dichroic mirror 7, the imaging lens 8, the mirror 9, and the objective lens 10 are shared with the observation means.

  The scanning unit 24 moves the irradiation position of the stimulation light on the sample surface in the X direction orthogonal to the optical axis and scans the sample 11 in the X direction, and the irradiation position of the stimulation light on the sample surface as the optical axis and A galvanometer mirror 24b that moves in the Y direction orthogonal to the X direction and scans the specimen 11 in the Y direction, and the galvanometer mirror 24a and the galvanometer mirror 24b are optically connected to the pupil plane 10P of the objective lens 10 in the middle. Are arranged such that a pupil conjugate plane conjugated to is formed. For example, the galvanometer mirror 24a and the galvanometer mirror 24b rotate about the Y axis and the X axis, respectively.

  The parallel plate 23 serving as a shift means is a parallel plate through which the stimulation light is transmitted, and the incident surface on which the stimulation light is incident can be inclined at an arbitrary angle with respect to the XY plane orthogonal to the optical axis. That is, the parallel plate 23 is configured to be rotatable about the X axis and the Y axis.

The control device 26 is connected to the laser light source 2, the scanning unit 5, the photodetector 15, the laser light source 16, the parallel plate 23, and the scanning unit 24, and controls the emission wavelength of the laser light source 2 and the laser light source 16. Transmission of scanning signals to the scanning means 5 and the scanning means 24, rotation control of the parallel plate 23, reception of electrical signals from the photodetector 15, and the like are performed.
The overall operation of the laser microscope 1 will be outlined with reference to FIG.

  The excitation light emitted as parallel light from the laser light source 2 is reflected by the dichroic mirror 4 incident through the shutter 3 and enters the scanning means 5. The scanning means 5 controlled by the scanning signal from the control device 26 deflects the excitation light in the X and Y directions orthogonal to the optical axis by the galvanometer mirror 5a and the galvanometer mirror 5b, respectively. The excitation light that has entered the pupil projection lens 6 from the scanning unit 5 is converged by the pupil projection lens 6, passes through the dichroic mirror 7 as convergent light or divergent light, and enters the imaging lens 8 as divergent light. The excitation light is converted again into parallel light by the imaging lens 8 and is condensed on the sample 11 by the objective lens 10 incident through the mirror 9. At the condensing position of the specimen 11 where the excitation light is condensed, the fluorescent substance contained in the specimen 11 is excited to emit fluorescence. The condensing position on the sample surface can be arbitrarily moved in the X direction and the Y direction by controlling the amount of deflection in the X direction and the Y direction by the scanning unit 5.

  The fluorescence generated from the specimen 11 is converted into parallel light by the objective lens 10 and travels in the opposite direction on the same optical path as the excitation light. That is, the light enters the dichroic mirror 4 via the mirror 9, the imaging lens 8, the dichroic mirror 7, the pupil projection lens 6, and the scanning unit 5. The fluorescence that has entered the dichroic mirror 4 passes through the dichroic mirror 4 and is collected by the confocal lens 12 onto a confocal stop 13 disposed on a plane conjugate with the sample surface (the front focal plane of the objective lens 10). The fluorescence generated from the focal position passes through the pinhole formed in the confocal stop 13, further passes through the barrier filter 14, and is detected by the photodetector 15.

  The photodetector 15 converts the detected fluorescence into an electrical signal and transmits it to the control device 26. The control device 26 generates an image of the sample 11 from the electrical signal from the photodetector 15 and the scanning signal transmitted to the scanning means 5 and displays it on a display device (not shown).

  On the other hand, the stimulation light emitted as parallel light from the laser light source 16 is reflected by the mirror 18 incident through the shutter 17 and enters the beam diameter changing optical system including the lens 19 and the lens 20. The stimulus light whose beam diameter is adjusted by the beam diameter changing optical system enters the field stop 21 as parallel light.

  The field stop 21 which is a variable stop is disposed on a surface optically conjugate with the sample surface. For this reason, by adjusting the aperture diameter of the field stop 21 and adjusting the beam diameter of the stimulation light that passes through the field stop 21, the irradiation of the stimulation light that irradiates the specimen 11 by defining the beam diameter of the stimulation light on the specimen surface. The range can be controlled. The intensity of the stimulation light has a Gaussian distribution, but the base part of the Gaussian distribution (the peripheral part of the light beam of the stimulation light) is blocked by the field stop 21, and a Gaussian distribution having a comparatively uniform intensity distribution is obtained. The central portion (the central portion of the luminous flux of the stimulation light) is selectively transmitted. Thereby, the nonuniformity of the intensity distribution of the stimulation light irradiated to the specimen 11 is suppressed, and the specimen 11 can be irradiated with a relatively uniform intensity.

  The stimulus light that has passed through the field stop 21 is converted into convergent light by the condenser lens 22 and passes through the parallel plate 23 that has entered as convergent light. The stimulation light transmitted through the parallel plate 23 is translated by the parallel plate 23 in the direction perpendicular to the optical axis by an amount determined by the refractive index of the parallel plate 23 and the angle of incidence on the parallel plate 23, and is scanned as a converged light beam 24. Is incident on. The amount by which the stimulation light moves in parallel with the optical axis on the parallel plate 23 (hereinafter referred to as a shift amount) is controlled by a control device 26 that drives the parallel plate 23.

  The scanning means 24 controlled by the scanning signal from the control device 26 deflects the stimulation light in the X direction and the Y direction orthogonal to the optical axis by the galvanometer mirror 24a and the galvanometer mirror 24b, respectively. Stimulation light deflected by the scanning unit 24 and incident on the pupil projection lens 25 as divergent light is converted into parallel light by the pupil projection lens 25 and applied to the dichroic mirror 7. Stimulation light incident on the dichroic mirror 7 is reflected by the dichroic mirror 7 and condensed by the imaging lens 8 through the mirror 9 onto the pupil plane 10P of the objective lens 10. The stimulation light collected on the pupil plane 10P is converted into parallel light again by the objective lens 10 and irradiated on the specimen 11. The irradiation position of the stimulation light on the sample surface can be arbitrarily moved in the X direction and the Y direction by controlling the deflection amount in the X direction and the Y direction by the scanning unit 24.

  FIG. 2 is a diagram for explaining a change in the condensing position of the stimulation light on the pupil conjugate plane due to the operation of the galvanometer mirror 24a. FIGS. The condensing position of the stimulation light in a state where the surface is inclined at different angles with respect to the optical axis is shown. FIG. 3 is a diagram for explaining the variation of the condensing position of the stimulation light on the pupil plane 10P of the objective lens 10. In FIG. FIG. 4 is a diagram for explaining the parallel movement of the stimulation light by the parallel plate 23. FIG. 5 is a diagram illustrating the condensing position of the stimulation light incident on the galvano mirror 24a via the parallel plate 23 whose rotation is controlled.

  The control of the parallel plate 23 and the scanning unit 24 for independently controlling the irradiation position and the irradiation angle of the stimulation light on the specimen surface will be described in detail with reference to FIGS.

First, the case where the parallel plate 23 is fixed at a predetermined angle with respect to the optical axis will be described.
As illustrated in FIG. 2, the scanning unit 24 is configured such that a pupil conjugate plane 24P is formed between the galvanometer mirror 24a and the galvanometer mirror 24b. Therefore, in the scanning unit 24, as illustrated in FIGS. 2A and 2B, the reflection surface of the galvano mirror 24a is inclined at a predetermined angle α with respect to the optical axis. When the stimulation light is collected on the optical axis of the pupil conjugate plane 24P (that is, the pupil conjugate position CP), the stimulus light is in the pupil conjugate plane 24P when the reflection surface of the galvano mirror 24a is inclined at a different angle β. The light is condensed at a position P1 deviated from the optical axis (hereinafter referred to as a condensing position).

  If the angle θ1 is an angle that is twice the rotation angle (= α−β) of the galvano mirror 24a from the state in which the stimulation light is collected at the pupil conjugate position CP (hereinafter referred to as the reference state), the stimulation light Is reflected by the galvano mirror 24a in a direction inclined by an angle θ1 with respect to the optical axis. Therefore, the distance h1 between the pupil conjugate position CP and the position P1 is calculated as D · tan θ1, where D is the distance between the deflection position of the stimulation light by the galvanometer mirror 24a and the pupil conjugate plane 24P.

  The deviation (distance h1) of the condensing position of the stimulation light from the pupil conjugate position CP on the pupil conjugate plane 24P is taken over by the pupil plane 10P of the objective lens 10. Therefore, as shown by the broken line in FIG. 3, the stimulus light is calculated by multiplying the distance h1 by the projection magnification of the pupil projection optical system including the pupil projection lens 25 and the imaging lens 8 even on the pupil plane 10P. The light is condensed at a position P2 deviated from the optical axis (pupil position PP) by h2. For this reason, the stimulation light does not enter the sample surface perpendicularly but enters the sample surface at the irradiation angle θ2. Here, the solid line in FIG. 3 shows the stimulation light when condensed at the pupil conjugate position CP, and shows a state in which the stimulation light is perpendicularly incident on the sample surface.

  The irradiation angle θ2 becomes larger as the condensing position (position P2) on the pupil plane 10P becomes farther from the optical axis. That is, as the amount of rotation of the galvanometer mirror 24a from the reference state increases, the irradiation angle on the sample surface increases. Therefore, when the parallel plate 23 is fixed, when the control device 26 transmits a scanning signal to the scanning unit 24 and changes the irradiation position of the stimulation light on the sample surface, the control unit 26 changes the position on the sample surface according to the irradiation position. The irradiation angle of the stimulus light changes.

Next, the case where the control device 26 controls the rotation of the parallel plate 23 according to the operation of the scanning unit 24 will be described.
The stimulation light passes through the parallel plate 23 and translates in a direction perpendicular to the optical axis, and the shift amount varies depending on the incident angle of the stimulation light as illustrated in FIG. Based on this, in the laser microscope 1 according to the present embodiment, as illustrated in FIG. 5, the control device 26 makes the condensing position of the stimulation light on the pupil conjugate plane 24P coincide with the pupil conjugate position CP. The parallel plate 23 is rotated according to the rotation amount of the galvanometer mirror 24a to control the shift amount. Thereby, the condensing position in the pupil plane 10P also coincides with the pupil position PP.

  When the control device 26 performs the above-described control, the angle of the principal ray on the axis incident on the pupil position PP changes according to the rotation amount of the galvanometer mirror 24a, and the irradiation position on the sample surface changes. Since the light is focused on the pupil position PP regardless of the amount of rotation, the irradiation angle on the specimen surface is maintained perpendicular to the specimen 11.

  Therefore, according to the laser microscope 1 according to the present embodiment, the control device 26 rotates the parallel plate 23 in accordance with the rotation amount from the reference state of the galvanometer mirror, that is, the irradiation position on the specimen surface, thereby changing the shift amount. By controlling, the stimulation light can irradiate the specimen 11 at a constant irradiation angle regardless of the irradiation position.

  For the sake of simplicity, the case where only the galvano mirror 24a operates has been described above, but the same method can be applied to the case where the galvano mirror 24b operates. When the galvano mirror 24b is operated, even if the stimulation light is condensed at the pupil conjugate position CP, the stimulation light is condensed at a position deviated from the pupil position PP by the rotation from the reference state of the galvano mirror 24b. There are things to do. This deviation can also be compensated by rotating the parallel plate 23. Specifically, the controller 26 controls the amount of shift in the X direction by controlling the amount of rotation of the parallel plate 23 around the Y axis in accordance with the rotation of the galvano mirror 24a, and according to the rotation of the galvano mirror 24b. The amount of shift in the Y direction may be controlled by controlling the amount of rotation of the parallel plate 23 around the X axis.

  In addition, the laser microscope 1 may include two parallel plates that rotate about the X axis and the Y axis, respectively, instead of the parallel plate 23 that rotates about the X axis and the Y axis.

  Further, the laser microscope 1 uses two mirrors (mirror 27a and mirror 27b) as illustrated in FIG. 6 instead of the parallel plate 23 that changes the shift amount by changing the incident angle of the stimulation light. The shift means 27 may be included. By moving the mirror 27b along the incident direction of the stimulation light, the stimulation light can be translated by the amount of movement.

  Further, the laser microscope 1 has four mirrors (mirror 28a, mirror 28b, mirror as illustrated in FIG. 7) instead of the parallel plate 23 that changes the shift amount by changing the incident angle of the stimulation light. 28c and a mirror 28d) may be included. The mirror 28b and the mirror 28c move together along the incident direction of the stimulation light incident on the shift means 28b, and the mirror 28d moves in the same direction as the movement of the mirror 28b and the mirror 28c. The stimulating light can be translated by the amount of movement of the mirror 28d by moving by an amount twice the amount of movement of 28c). The shift means 28 is preferable to the shift means 27 in that the stimulation light can be translated without changing the optical path length of the stimulation light.

  Further, in the laser microscope 1, due to chromatic aberration, when the wavelength of the stimulation light emitted from the laser light source 16 is different, the condensing position of the stimulation light on the pupil plane 10P also changes. For this reason, it is desirable that the control device 26 controls the shift amount according to the irradiation position and the wavelength of the stimulation light.

  FIG. 8 is a diagram illustrating a part of the configuration of the laser microscope according to the present embodiment. The laser microscope illustrated in FIG. 8 irradiates the specimen 33 with laser light at an angle greater than the critical angle, thereby exciting the specimen 33 with evanescent light that oozes from the total reflection surface (hereinafter referred to as TIRF). This is referred to as a microscope.).

  The TIRF microscope 29 deviates the laser beam incident through the imaging lens 30 and the mirror 31 from the optical axis of the pupil plane 32P of the objective lens 32 in order to irradiate the specimen 33 at a predetermined irradiation angle equal to or greater than the critical angle. The point of condensing at the position P3 is different from the laser microscope 1 according to the first embodiment. Other points are the same as those of the laser microscope 1 according to the first embodiment.

In the TIRF microscope 29, similarly to the laser microscope 1 according to the first embodiment, a control device (not shown) controls the shift amount by the shift unit according to the driving of the scanning unit, that is, the irradiation position on the specimen surface. The condensing position of the laser light on the pupil plane 32P can be maintained at the position P3 deviated from the optical axis.
Therefore, even with the TIRF microscope 29 according to the present embodiment, it is possible to irradiate the specimen 33 with the laser beam at a constant irradiation angle regardless of the irradiation position.

  In the control device of the TIRF microscope 29, it is desirable to control the shift amount according to the wavelength of the laser light in addition to the irradiation position, similarly to the control device 26 of the laser microscope 1 according to the first embodiment.

In the TIRF microscope 29, as described above, the laser light is condensed at a position deviating from the optical axis on the pupil plane 32P. However, depending on the objective lens, the diameter of the pupil differs, so that the laser is formed around or outside the pupil of the objective lens 32. When the light is condensed, vignetting may occur and the uniformity of the light quantity and intensity distribution of the laser light irradiated on the specimen 33 may be reduced. For this reason, it is desirable that the control device of the TIRF microscope 29 controls the shift amount according to the pupil diameter of the objective lens in addition to the irradiation position.
When changing the observation magnification while keeping the irradiation angle constant, it is desirable to control the shift amount according to the change in the magnification of the objective lens.

  As described above, in the first and second embodiments, the control device drives the scanning unit to change the irradiation position on the sample surface, and drives the shift unit to change the irradiation angle on the sample surface by driving the scanning unit. However, the control by the control device is not limited to such control. The control device may positively change the irradiation angle instead of compensating for the change in the irradiation angle due to the driving of the scanning unit. Thus, the sample may be irradiated with laser light at an arbitrary irradiation angle regardless of the irradiation position.

DESCRIPTION OF SYMBOLS 1 ... Laser microscope 2, 16 ... Laser light source 3, 17 ... Shutter 4, 6, ... Dichroic mirror 5, 24 ... Scanning means 5a, 5b, 24a, 24b ... Galvano mirror 6, 25 ... pupil projection lenses 8, 30 ... imaging lenses 9, 18, 27a, 27b, 28a, 28b, 28c, 28d, 31 ... mirrors 10, 32 ... objective lenses 10P, 32P ... Pupils 11, 33 ... Sample 12 ... Confocal lens 13 ... Confocal stop 14 ... Barrier filter 15 ... Photo detectors 19, 20 ... Lens 21 ... Field stop 22 ... Condensing lens 23 ... Parallel plate 24P ... Pupil conjugate surface 26 ... Control devices 27, 28 ... Shift means 29 ... TIRF microscope CP ... Pupil conjugate position PP ... Pupil positions P1, P2, 3 ... position

Claims (9)

A laser light source for emitting laser light;
An objective lens for irradiating the specimen with the laser beam;
A condensing lens for condensing the laser light on a pupil conjugate plane conjugate with the pupil plane of the objective lens;
Scanning means for scanning the specimen by moving an irradiation position for irradiating the specimen with the laser beam;
Shift means for moving the laser light in a direction perpendicular to the optical axis;
And a shift control unit that controls the shift amount by which the laser beam moves in parallel by driving the shift unit. The laser microscope according to claim 1,
The scanning means includes
First scanning means for scanning the specimen in a first direction orthogonal to the optical axis;
Second scanning means for scanning the specimen in a second direction perpendicular to the optical axis and perpendicular to the first direction,
The laser microscope characterized in that the shift control means controls the shift amount according to the irradiation position. The laser microscope according to claim 2,
The laser microscope characterized in that the shift control means controls the shift amount so that the laser beam irradiates the specimen at a fixed irradiation angle regardless of the irradiation position. In the laser microscope according to claim 3,
The first scanning means is a first mirror that rotates around a first rotation axis orthogonal to the optical axis,
The second scanning means is a second mirror that rotates around a second rotation axis that is orthogonal to the optical axis and orthogonal to the first rotation axis,
The laser microscope, wherein the pupil conjugate plane is located between the first mirror and the second mirror. In the laser microscope according to claim 3,
The laser microscope, wherein the shift control unit controls the shift amount so that a condensing position of the laser light on the pupil plane is located on an optical axis. In the laser microscope according to claim 3,
The laser microscope characterized in that the shift control means controls the shift amount so that the condensing position of the laser light on the pupil plane is located at a position deviating from the optical axis. In the laser microscope according to claim 3,
The laser microscope, wherein the shift control unit controls the shift amount according to a magnification of the objective lens. In the laser microscope according to claim 3,
The shift control means controls the shift amount according to the wavelength of the laser light. In the laser microscope according to claim 3,
The shift means is a parallel plate through which the laser light is transmitted,
The laser microscope, wherein the shift control means rotates the shift means around a rotation axis orthogonal to the optical axis.