JP5307373B2 - Optical scanning microscope - Google Patents

Description

  The present invention relates to an optical scanning microscope.

Conventionally, a microscope is known in which an optical element having a positive refractive power and a negative refractive power is arranged between a light source and an objective lens, and the working distance of the objective lens is changed by changing a physical distance (for example, Patent Documents). 1).
In addition, a microscope is also disclosed in which an adapter lens can be attached to and detached from a finite objective lens (see, for example, Patent Document 2). According to this, the working distance of the objective lens is changed by combining the small-diameter finite objective and the adapter lens and moving the adapter lens in the optical axis direction.

  A microscope in which a finite distance objective lens is detachable is also known (for example, see Patent Document 3). According to this, the working distance of the objective lens is changed by combining the finite objective and the imaging lens and moving the imaging lens in the optical axis direction.

JP-A-2005-70477 JP 2006-79000 A JP 2006-139181 A

  However, in the microscopes disclosed in these Patent Documents 1 to 3, nothing is described regarding the positional relationship between the objective lens and the imaging lens and the optical system that changes the focus, and the position of the optical system is not described. If the relationship is not taken into consideration, there is a disadvantage that the observation magnification greatly changes when the working distance of the objective lens is changed by moving the lens constituting the optical system in the optical axis direction.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a focus adjustment unit and an optical scanning microscope that do not change the observation magnification so much even if the working distance of the objective lens is changed. .

In order to achieve the above object, the present invention provides the following means.
The present invention provides an objective optical system that converts light from a sample into substantially parallel light, an imaging optical system that forms an image of substantially parallel light from the objective optical system at a predetermined position, and a predetermined optical system using the imaging optical system. A pupil projection optical system for converting the light imaged at the position to a substantially parallel light beam, and a lateral scanning means for scanning the observation position of the sample in the horizontal direction by deflecting the angle of the substantially parallel light beam from the pupil projection optical system And driving the objective optical by driving in the optical axis direction at least one of a photodetector that detects light from the sample that has passed through the lateral scanning means, and the imaging optical system or the pupil projection optical system. A lens driving means for changing the working distance of the system, and the imaging optical system is arranged such that the front side focal point is positioned in the vicinity of the rear focal point of the objective optical system with the sample side as the front side. An optical scanning microscope is provided.

According to the present invention, the working distance of the objective lens is changed by driving at least one of the inner field of the imaging optical system or the pupil projection optical system in the optical axis direction. In this case, the magnification when moving either the imaging optical system or the pupil projection optical system in the optical axis direction by placing the front focal point of the imaging optical system in the vicinity of the rear focal point of the objective optical system. The change of can be suppressed.
Here, the substantially parallel light includes not only strictly parallel light but also divergent light and convergent light at a gentle angle.

In the above invention, it is preferable that the lens driving unit drives the imaging optical system in the optical axis direction, the pupil projection optical system is fixed, and satisfies the following expression (1).
| D1a × Δs | / (Ftl) ² ≦ 0.05 (1)
Here, Δs: total moving distance of the imaging optical system, D1a: a rear focal point of the objective optical system and a front focal point of the imaging optical system when the imaging optical system moves to an intermediate position , Ftl: the focal length of the imaging optical system.
By doing in this way, the change of the magnification when the working distance is changed can be reduced.

In the above invention, D1a = 0 is preferable.
By doing in this way, the change of the magnification when the working distance is changed can be eliminated.

In the above invention, the lens driving unit may drive the pupil projection optical system in the optical axis direction, the imaging optical system may be fixed, and the following conditional expression (2) may be satisfied.
| D1 × δs | / (Ftl) ² ≦ 0.05 (2)
Where δs: total moving distance of the pupil projection optical system, D1: distance between the rear focal point of the objective optical system and the front focal point of the imaging optical system, Ftl: focal distance of the imaging optical system. .
By doing in this way, the change of the magnification when the working distance is changed can be reduced.

In the above invention, D1 = 0 is preferable.
By doing in this way, the change of the magnification when the working distance is changed can be eliminated.

In the above invention, at least one of the pupil projection optical system and the imaging optical system may be moved around a position that is established as an afocal optical system.
By doing in this way, the change of the magnification when the working distance is changed can be further suppressed.

In the above invention, the lateral scanning means may be arranged near the rear focal point of the pupil projection optical system.
By doing in this way, the sample side can be made close to a telecentric optical system (exit pupil is infinite).

In the above invention, the light emitting unit that emits light for illuminating or exciting the sample, the light receiving unit that receives light from the sample guided toward the photodetector, and the light emitting unit from the light emitting unit A first collimating optical system that converts light into substantially parallel light; a second collimating optical system that collects light from the sample onto the light receiving unit; light from the light emitting unit and light from the sample; A scanning optical system comprising a detection light separating means that separates the horizontal scanning means, and a pupil position in the vicinity of the horizontal scanning means by moving the scanning optical system in the optical axis direction of the pupil projection optical system. It is good also as providing the pupil position adjustment means to make it correspond.
Furthermore, in the said invention, it is good also as observing in the state which contact | adhered the front-end | tip of the said objective optical system to the said sample.

  According to the present invention, there is an effect that the working distance of the objective lens can be changed without significantly changing the observation magnification.

Hereinafter, a laser scanning microscope (optical scanning microscope) 1 according to a first embodiment of the present invention will be described with reference to FIGS.
A laser scanning microscope 1 according to the present embodiment includes a laser light source 2 that emits laser light, a coupling optical system 3 that condenses the laser light from the laser light source 2, and light that is condensed by the coupling optical system 3. Detected by the optical fiber 4 that guides the laser beam, the microscope main body 5 connected to the laser light source by the optical fiber 4, the detection optical system 15, the control unit 16 that controls these, and the detection optical system 15. And a display unit (not shown) for displaying an image of the reflected fluorescence or reflected light.

  The microscope body 5 includes a collimating optical system 6 that converts laser light emitted from the optical fiber 4 into substantially parallel light, a dichroic mirror 7 that deflects laser light that has been converted into substantially parallel light by the collimating optical system 6, A proximity galvanometer mirror 8 that two-dimensionally scans the laser beam deflected by the dichroic mirror 7, a pupil projection optical system 9 that collects the laser beam scanned by the proximity galvanometer mirror 8, and the collected light as a sample The image forming optical system 10 and the objective optical system 11 for condensing light onto A and the lens driving means 12 for driving the image forming optical system 10 in the optical axis direction are provided.

The microscope body 5 includes a dichroic mirror 7 that transmits fluorescence or reflected light from the sample A that has returned through the objective optical system 11, the imaging optical system 10, the pupil projection optical system 9, and the proximity galvanometer mirror 8. A coupling optical system 13 that condenses the light on the optical fiber 14 and an optical fiber 14 that guides fluorescence or reflected light from the sample A collected by the coupling optical system 13 are provided.
The microscope body 5 is provided so as to be movable in directions of three axes (XYZ) orthogonal to each other, and is provided so as to be rotatable around each axis, and arbitrarily adjusts the position and angle of the tip of the objective optical system 11. Be able to.

The proximity galvanometer mirror 8 scans the observation position in a direction substantially perpendicular to the optical axis of the objective optical system 11. The intensity distribution of light from the sample A in a range corresponding to the swing angle of the proximity galvanometer mirror 8 is displayed on the display unit.
The objective optical system 11 is configured to suppress blurring of an observation image due to respiration or pulsation of the sample A by bringing its tip into close contact with the sample A.

In this case, it is desirable that the condition of the following expression (1) is satisfied.
| D1a × Δs | / (Fla) ² ≦ 0.05 (1)
Here, Δs: total moving distance of the imaging optical system 10, D1a: a rear focal point of the objective optical system 11 and a front focal point of the imaging optical system 10 when the imaging optical system 10 moves to an intermediate position , Fla: the focal length of the imaging optical system 10.

  The detection optical system 15 includes a collimating optical system 17 that converts the fluorescence or reflected light guided by the optical fiber 14 into substantially parallel light, a plurality of dichroic mirrors 18 and mirrors 19 that branch for each wavelength, and a barrier filter 20. The condenser lens 21 and the photodetector 22 are provided. In the figure, reference numeral 23 denotes a dichroic mirror, and reference numeral 24 denotes a mirror.

The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described.
In order to observe the sample A using the laser scanning microscope 1 according to the present embodiment, the microscope is connected from the laser light source 2 through the optical fiber 4 with the tip of the objective optical system 11 in close contact with the sample A. The laser beam guided to the main body 5 is deflected by the dichroic mirror 7, scanned two-dimensionally by the proximity galvanometer mirror 8, and applied to the sample A via the pupil projection optical system 9, the imaging optical system 10, and the objective optical system 11. Condensate.

  In the sample A irradiated with the laser light, fluorescence is generated by exciting the fluorescent material, and the generated fluorescence is converted into divergent light or convergent light of a gentle angle including parallel light by the objective optical system 11, After being imaged by the imaging optical system 10 and made substantially parallel light by the pupil projection optical system 9, the light returns through the proximity galvanometer mirror 8, passes through the dichroic mirror 7, and is coupled to the optical fiber 14 by the coupling optical system 13. After being condensed at the end and guided by the optical fiber 14, it is detected by the detection optical system 15.

In this case, for example, as shown in FIG. 4, when the imaging optical system 10 is moved in the optical axis direction by the lens driving unit 12, the working distance of the objective optical system 11 (focusing on the tip of the objective optical system as a reference). Distance) changes.
Therefore, an image having an arbitrary depth in the sample A can be observed without moving the objective optical system 11. Furthermore, if a plurality of images are acquired while moving the imaging optical system 10, a three-dimensional image of the sample A can be acquired.

Here, the principle of the present invention will be described with reference to FIGS.
FIG. 2 shows a reference position of each optical system, and FIG. 3 shows a state where the optical system is moved from the reference position.
As for the reference position, the imaging optical system 10 and the pupil projection optical system 9 become an afocal optical system (that is, the rear focal point of the imaging optical system 10 and the front focal point of the pupil projection optical system 9 coincide). Furthermore, the position of the objective optical system 11 is set to a position where the distance between the rear focal point of the objective optical system 11 and the front focal point of the imaging optical system 10 is D1.

2 and 3, the points P1 to P8 are as follows.
P1: the front focal point of the objective optical system 11;
P2: position of the objective optical system 11,
P3: the rear focal point of the objective optical system 11,
P4: the front focal point of the imaging optical system 10,
P5: the position of the imaging optical system 10,
P6: rear focus of the imaging optical system 10 and front focus of the pupil projection optical system 9 P7: position of the pupil projection optical system 9;
P8: Rear focus of pupil projection optical system 9.

When the rear side of the pupil projection optical system 9 is a substantially parallel light beam,
Zwd = n (Δ−δ) (Fob / Ftl) ² / (1 + A) (3)
The ratio of the height Job in the direction perpendicular to the optical axis of the imaging position on the sample A side when the parallel light beam on the rear side of the pupil projection optical system 9 is at an angle θpl with respect to the optical axis, that is, (θpl / Yob )
(Θpl / Yob) = M / Fpl (4)
However,
M = − (Ftl / Fob) (1 + A) (5)
A = (D1 + Δ) (Δ−δ) / Ftl ² (6)
Zwd: change amount of the working distance of the objective optical system 11 from the reference position P1,
Δ: Displacement amount from the reference position P5 of the imaging optical system 10 δ: Displacement amount from the reference position P7 of the pupil projection optical system 9 n: Refractive index Fob on the sample side: Focal length Ftl of the objective optical system 11: Image formation Focal length Fpl of the optical system 10: Focal length M of the pupil projection optical system 9: Horizontal magnification θpl when the sample is imaged by the objective optical system 11 and the imaging optical system 10: pupil projection when the ray height of the object is Job An angle with respect to the optical axis of the parallel light behind the optical system 9 (corresponding to a half value of the angle of the proximity galvanometer mirror 8).

Also, the exit pupil position (when the objective-side entrance pupil is −∞) Zp is
Zp = {δ− (Fpl / Ftl) ² · (D1 + Δ) / (1 + A)} (7)
It becomes.

Therefore, in the laser scanning microscope according to the present embodiment, as shown in FIG. 4, the pupil projection optical system 9 is fixed and the imaging optical system 10 is driven in the optical axis direction by the lens driving unit 12. δ = 0, and from the above formulas (3) to (6),
Zwd = n × Δ (Fob / Ftl) ² / (1 + Atl) (3 ′)
The relationship holds.
here,
(Θpl / Yob) = Mtl / Fpl (4 ′)
However,
Mtl = − (Ftl / Fob) (1 + Atl) (5 ′)
Atl = (D1 + Δ) × Δ / Ftl ² (6 ′)
It is.

From equation (3 ′), it can be seen that the range in which Zwd changes is mainly the range in which Δ moves.
Here, the moving range of the imaging optical system is moved in the vicinity of Δ = 0 (position where the pupil optical system and the imaging optical system become afocal), and the rear focal point of the objective optical system is It is desirable to arrange each optical system in the vicinity of the front focal point, that is, to reduce D1.
This reduces the amount of change of Atl with respect to Δ near Δ = 0 from equation (6 ′). Therefore, even if the imaging optical system is moved (even if Δ is changed), the lateral magnification Mtl, the ray angle, and the object height The change in the ratio (θpl / Yob) can be reduced.
Specifically, it is desirable to set D1a so as to satisfy the above formula (1). Under this condition, changes in Atl and lateral magnification Mtl can be suppressed to approximately 5% or less.

Example 1
5 shows Δ (displacement of the imaging optical system) on the horizontal axis and the vertical axis shows Zwd (change in working distance WD), and FIG. 6 shows Δ on the horizontal axis and (θpl / Yob), that is, horizontal magnification M. The amount proportional to is shown on the vertical axis.
For example, the case where Fob = 9 mm, Ftl = 50 mm, Fpl = 12 mm, Δ: −1.5 mm to +1.5 mm, ie, Δs = 3 mm, D1a = 15 mm is shown by the solid line (A) in FIGS. .

If this is applied to equation (1),
| D1a × Δs | / (Ftl) ² = 0.018 ≦ 0.05 (1)
Thus, the above formula (1) is satisfied.
At this time, the change in Atl is only 0.0018 in the movable range of the imaging optical system. As described above, when the expression (1) is satisfied, the change in the value of Atl in (6 ′) is reduced over the entire range in which the imaging lens is moved. Therefore, even if the working distance WD changes, The change in the magnification Mtl and the change in the ratio (4 ′) of the ray angle to the object height (θpl / Yob) become small enough for practical use.

Furthermore, if it is desired to make the change in lateral magnification very small, D1a = 0 may be set. Fob, Ftl, Fpl, and Δ indicate the behavior when D1 is set to 0 under the same conditions as described above, with broken lines (B) in FIGS.
In this case, since the slope of Atl with respect to Δ becomes 0 near Δ = 0, the change in the lateral magnification becomes very small. In this embodiment, the change in Atl is 0.0009 over the entire region.

Next, a laser scanning microscope 30 according to the second embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, portions having the same configuration as those of the laser scanning microscope 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

In the laser scanning microscope 30 according to the present embodiment, the laser scanning microscope 1 according to the first embodiment drives the imaging optical system 10 in the optical axis direction by the lens driving unit 12, whereas As shown in FIG. 7, the difference is that the pupil projection optical system 9 is driven in the optical axis direction.
Since the working distance is changed by driving the pupil projection optical system 9 in the optical axis direction, an image of an arbitrary depth in the sample A can be observed.

Therefore, in the laser scanning microscope 30 according to the present embodiment, as shown in FIG. 8, the imaging optical system 10 is fixed and the pupil projection optical system 9 is driven in the optical axis direction by the lens driving means 12. , Δ = 0, and Zwd = −n × δ (Fob / Ftl) ² / (1 + Apl) (3 ″) from the above equations (3) to (6).
The relationship holds.
here,
(Θpl / Yob) = Mpl / Fpl (4 ″)
However,
Mpl = − (Ftl / Fob) (1 + Apl) (5 ″)
Ap1 = −δ × D1 / Ftl ² (6 ″)
It is.

From equation (3 ″), it can be seen that the range in which Zwd changes is mainly the range in which Δ moves.
Here, it is desirable to make the rear focal point of the objective optical system 11 near the front focal point of the imaging optical system 10, that is, to reduce D1. When D1 is small, even if the pupil projection optical system is moved (δ is changed), the change in the lateral magnification Mpl and the ratio between the ray angle and the object height (θpl / Yob) can be reduced.

From the equation (6 ″), it can be seen that the change in Ap1 is proportional to D1 and δ.
Since the range in which Zwd changes can be mainly the range in which δ moves, the range of δ does not change greatly. Therefore, the change in Mpl is mainly determined by D1.
Specifically, for the total movement amount δs of the pupil projection lens,
| D1 × δs | / Ftl ² ≦ 0.05 (2)
Is desirable.
Under this condition, the change in Ap1 can be suppressed to 0.05 or less.

(Example 2)
9 shows δ (displacement of the pupil projection optical system) on the horizontal axis and the vertical axis shows Zwd (change in working distance WD), and FIG. 10 shows δ on the horizontal axis and (θpl / Yob), that is, horizontal magnification. The amount proportional to is shown on the vertical axis.
For example, the case where Fob = 9 mm, Ftl = 50 mm, Fpl = 12 mm, δ: −1.5 mm to +1.5 mm, that is, δs = 3 mm and D1a = 15 mm is shown by solid lines (A) in FIGS. .

If this is applied to condition (2),
| D1 × δs | / Ftl ² = 0.018 ≦ 0.05
Therefore, the above formula (2) is satisfied.
At this time, the change in ApI is only 0.0018. Thus, when the expression (2) is satisfied, the change in the value of Ap1 in the expression (6 ″) becomes small. Therefore, even if the working distance WD changes, the change in the lateral magnification Mpl in the expression (5 ″) and Formula (4 ″)
The change in the ratio of the light beam angle to the object height (θpl / Yob) becomes small enough for practical use. "

Furthermore, if it is desired to make the change in lateral magnification very small, D1 = 0 may be set. Fob, Ftl, Fpl, and Δ indicate the behavior when D1 is set to 0 under the same conditions as described above with broken lines (B) in FIGS.
In this case, since Ap1 is always Ap = 0 regardless of the value of δ, the lateral magnification does not change at all.

Next, a laser scanning microscope 40 according to a third embodiment of the present invention will be described below with reference to FIGS. 11 and 12.
In the description of the present embodiment, portions having the same configuration as those of the laser scanning microscope 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  In the laser scanning microscope 40 according to the present embodiment, in addition to the laser scanning microscope 1 according to the second embodiment driving the pupil projection optical system 9 in the optical axis direction by the lens driving means 12. 11, the emission end (light emission part) 4a of the optical fiber 4, the incident end (light receiving part) 14a of the optical fiber 14, the collimating optical system 6, the coupling optical system 13, A scan optical system 41 including a dichroic mirror 7 and a proximity galvanometer mirror 8 and a pupil position adjusting means 42 for moving the scan optical system 41 integrally in the optical axis direction of the pupil projection optical system 9 are provided.

By operating the pupil position adjusting means 42 and moving the scan optical system 41 integrally in the optical axis direction of the pupil projection optical system 9, the pupil position can be matched with the proximity galvanometer mirror 8.
The adjustment of the exit pupil position Zp by the pupil position adjusting means 42 may be performed according to the equation (7). Here, Zp = 0 coincides with the rear focal position of the pupil projection optical system 9 at the reference position shown in FIG.

As shown in FIG. 11, when the imaging optical system 10 is fixed and the pupil projection optical system 9 is moved in the optical axis direction, the equation (7) is modified as follows.
Zp = {δ− (Fpl / Ftl) ² · D1 / (1 + A)}
If Equation (1) or Equation (2) holds, 1 >> A,
Zp = δ− (Fpl / Ftl) ² · D1, and as shown in FIG. 12, the position Zp of the exit pupil is linearly slid by substantially the same amount δ with respect to the displacement δ of the pupil projection optical system 9. That's fine.

In this embodiment, the case where the pupil projection optical system 9 is driven by the lens driving unit 12 has been described. Instead, the pupil projection optical system 9 is fixed and the imaging optical system 10 is moved in the optical axis direction. When moving, equation (7) is modified as follows.
Zp = − (Fpl / Ftl) ² · (D1 + Δ)
Accordingly, as shown in FIG. 12, the position Zp of the exit pupil may be linearly slid by − (Fpl / Ftl) ² · Δ with respect to the displacement Δ of the imaging optical system 10.

  As shown in FIG. 13, if the lens driving means 12 and 12 'are provided in both the pupil projection optical system 9 and the imaging optical system 10, the objective optical system depends on the type of the objective optical system 11 to be attached. Even in the case where the rear focal point of the objective optical system 11 is different with respect to the 11 barrel positions, it can be easily adjusted. That is, first, the lens driving unit 12 'is driven to make the front focal point of the imaging optical system 10 coincide with the rear focal point of the objective optical system 11, and then the pupil projection optical system 9 is moved in the optical axis direction by the lens driving unit 12. To adjust the working distance of the objective optical system 11, and finally, the pupil position adjusting means 42 is operated to move the scan optical system 41 to adjust the pupil position.

1 is an overall configuration diagram showing a laser scanning microscope according to a first embodiment of the present invention. It is a figure explaining the principle of the displacement of an optical system and the change of a working distance in the laser scanning microscope of FIG. 1, and is a figure by which each optical system is arrange | positioned in the reference position. It is a figure which shows the state which each optical system displaced from the reference position of FIG. In the laser scanning microscope of FIG. 1, it is a figure explaining the change of the working distance when only an imaging optical system is displaced. 6 is a graph showing the relationship between the displacement of the imaging optical system and the amount of change in working distance in the laser scanning microscope of FIG. 6 is a graph showing the relationship between the displacement of the imaging optical system and the lateral magnification in the laser scanning microscope of FIG. It is a whole block diagram which shows the laser scanning microscope which concerns on the 2nd Embodiment of this invention. In the laser scanning microscope of FIG. 7, it is a figure explaining the change of a working distance when only a pupil projection optical system has displaced. It is a graph which shows the relationship between the displacement of the pupil projection optical system in the laser scanning microscope of FIG. 8, and the variation | change_quantity of a working distance. It is a graph which shows the relationship between the displacement of a pupil projection optical system in the laser scanning microscope of FIG. 8, and lateral magnification. It is a whole block diagram which shows the laser scanning microscope which concerns on the 3rd Embodiment of this invention. 12 is a graph showing the displacement of the exit pupil position with respect to the displacement of the imaging optical system or pupil projection optical system in the laser scanning microscope of FIG. FIG. 12 is a general configuration diagram showing a laser scanning microscope in which an imaging optical system, a pupil projection optical system, and a scanning optical system are movably provided as a modification of the laser scanning microscope of FIG. 11.

Explanation of symbols

A Sample 1,30,40 Laser scanning microscope (optical scanning microscope)
4a Emission end (light emission part)
6 Collimating optical system (first collimating optical system)
7 Dichroic mirror (detection light separation means)
8 Proximity galvanometer mirror (lateral scanning means)
DESCRIPTION OF SYMBOLS 9 Pupil projection optical system 10 Imaging optical system 11 Objective optical system 12,12 'Lens drive means 13 Coupling optical system (2nd collimating optical system)
14a Incident end (light receiving part)
41 Scanning optical system 42 Pupil position adjusting means

Claims (9)

An objective optical system that converts light from the sample into substantially parallel light;
An imaging optical system that forms substantially parallel light from the objective optical system at a predetermined position;
A pupil projection optical system that converts light imaged at a predetermined position by the imaging optical system into a substantially parallel light beam;
A lateral scanning means for deflecting the angle of the substantially parallel light beam from the pupil projection optical system and scanning the observation position of the sample laterally;
A photodetector for detecting light from the sample via the lateral scanning means;
Lens driving means for changing the working distance of the objective optical system by driving at least one of the imaging optical system or the pupil projection optical system in the optical axis direction;
The imaging optical system is an optical scanning microscope in which the sample side is the front side and the front focal point is positioned in the vicinity of the rear focal point of the objective optical system. The lens driving means drives the imaging optical system in the optical axis direction;
The pupil projection optical system is fixed;
The optical scanning microscope of Claim 1 which satisfy | fills the following formula | equation (1).
| D1a × Δs | / (Ftl) ² ≦ 0.05 (1)
here,
Δs: total moving distance of the imaging optical system,
D1a: an interval between the rear focal point of the objective optical system and the front focal point of the imaging optical system when the imaging optical system moves to an intermediate position;
Ftl: focal length of the imaging optical system. D1a = 0
The optical scanning microscope according to claim 2. The lens driving means drives the pupil projection optical system in the optical axis direction;
The imaging optical system is fixed;
The optical scanning microscope according to claim 1, wherein the following conditional expression (2) is satisfied.
| D1 × δs | / (Ftl) ² ≦ 0.05 (2)
here,
δs: total moving distance of the pupil projection optical system,
D1: an interval between the rear focal point of the objective optical system and the front focal point of the imaging optical system;
Ftl: focal length of the imaging optical system. D1 = 0
The optical scanning microscope according to claim 4.   The optical scanning microscope according to any one of claims 1 to 5, wherein at least one of the pupil projection optical system or the imaging optical system is moved around a position where the pupil projection optical system and the imaging optical system are established as an afocal optical system.   The optical scanning microscope according to any one of claims 1 to 6, wherein the lateral scanning unit is disposed in the vicinity of a rear focal point of the pupil projection optical system. A light emitting part for emitting light for illuminating or exciting the sample;
A light receiving portion for receiving light from the sample guided toward the photodetector;
A first collimating optical system that makes light from the light emitting section substantially parallel light;
A second collimating optical system for condensing light from the sample onto the light receiving unit;
Detection light separating means for separating light from the light emitting section and light from the sample;
A scanning optical system comprising the lateral scanning means;
The pupil position adjusting means for moving the scanning optical system in the optical axis direction of the pupil projection optical system to match the pupil position in the vicinity of the lateral scanning means. Optical scanning microscope.   The optical scanning microscope according to any one of claims 1 to 8, wherein observation is performed in a state in which a tip of the objective optical system is in close contact with the sample.

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