JP2002148521A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope which makes a sample surface observable without being affected by the regular reflected light of illumination light. SOLUTION: The parallel luminous fluxes released from a laser beam source 1 are reflected by a scan mirror 11 so as to be changed in their direction by 90 deg. with a dichroic mirror 4 and are limited of an aperture angle by an aperture angle limiting aperture 10, following which the luminous fluxes are reflected by a scan mirror 11. The luminous fluxes focus at the surface of the sample 7 and form a spot through an objective lens 6. The scan mirror 11 is made turnable around the axis of turning in a two-dimensional direction and is adapted to prevent only the position at one point of its reflecting surface from being changed by turning. The scan mirror 11 is so arranged that the axis of turning thereof exists at a position apart from the optical axis of the objective lens 6 on the focal plane of the objective lens 6. As a result, the regular reflected light is no more detected by a photoelectric detector 24 and noise is lowered.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for irradiating a sample surface with light and generating reflected light from the sample surface or light generated from the sample surface.
The present invention relates to a microscope for detecting and observing by an imaging device or a photoelectric detector.

[0002]

2. Description of the Related Art A confocal microscope is known as a microscope which irradiates a sample surface with light and detects reflected light from the sample surface or light generated from the sample surface by an imaging device or a photoelectric detector and observes the same. It is known. This is, for example, J.
HANDBOOK OF BIOLOGICAL CONFOCAL MICR edited by B. Powley
OSCOPY (SECOND ED.PLENUM PRESS, NEW YORK, 1995), P1
41, FIGURE3 and FIGURE4.

FIG. 6 shows an outline of the optical system. The laser light emitted from the laser light source 21 is converted into a parallel light beam through the beam expander 22 and is reflected by the dichroic mirror 23 to change the direction by 90 °. Then, after being reflected by the scan mirrors 24 and 25, the light passes through the scan lens 26 and is condensed on the surface of the sample 28 by the objective lens 27 to form a spot.

The light reflected from the surface of the sample 28 or the sample 2
The fluorescent light from 8 reaches the dichroic mirror 23 through the same path as the illumination light in the opposite direction, and the reflected light having the same wavelength as the illumination light is reflected, and the fluorescent light having a different wavelength from the illumination light passes through the dichroic mirror 23. The light is condensed on the imaging surface 30 of the imaging device by the detection lens 29.

As is apparent from the figure, since the surface of the sample 28 and the imaging surface 30 of the imaging device are conjugate, the sample 2
An image of the fluorescence generated from the position corresponding to the eight spots is formed on the imaging surface 30 of the imaging device. Scan mirror 2
4 and 25, when the optical axis of the objective lens 27 is the z-axis,
The position of the spot formed on the surface of the sample 28 is changed in the x-axis direction and the y-axis direction. By driving these scan mirrors 24 and 25, the images of the fluorescent light generated from different positions on the surface of the sample 28 are changed. Is formed on the imaging surface 30 of the imaging device, whereby a fluorescent image of the surface of the sample 28 can be captured. What has been described above is a so-called fluorescence microscope, but it is also possible to capture an image of light reflected from the surface of the sample 28 by changing the dichroic mirror 23 to a half mirror.

[0006]

In the optical system as shown in FIG. 6, the optical axis of the illumination light coincides with the optical axis of the detected light as described above, and the optical axis is perpendicular to the sample surface. ing. That is, the illumination light is emitted from the objective lens 2 perpendicular to the sample surface.
The light condensed on the surface of the sample 28 having an optical axis parallel to the optical axis of the light source 7 and emitted or reflected from the surface of the sample 28 also has the same optical axis as the illumination light. Image.

However, when a sample is to be observed with a fluorescence microscope having such an optical system, if the sample is placed on a glass surface having a high reflectance, both the specular reflection light and the fluorescence are used as objectives. The light is guided to the dichroic mirror 23 by the lens. The dichroic mirror 23 is selected so as to transmit the fluorescence and reflect the reflected light due to the difference between the wavelengths of the reflected light and the fluorescent light. However, it is difficult to completely separate the two from each other.
It is inevitable that a certain amount of reflected light is mixed in the light reaching zero. Therefore, there is a problem that reflected light becomes detection noise and hinders fluorescence observation.

[0008] Even when not using a fluorescence microscope, when observing the surface state of a sample having a high reflectivity, the intensity of specularly reflected light is high, thereby hiding light information indicating the surface state of the sample. In some cases, accurate observations cannot be made.

The present invention has been made in view of such circumstances, and the specular reflection light of illumination light ("specular reflection light" in the present specification is defined assuming that the sample surface is a perfect mirror surface). It is an object of the present invention to provide a microscope capable of observing the surface of a sample without being affected by the reflected light, in particular, a fluorescence microscope.

[0010]

A first means for solving the above problems is to irradiate a sample surface with an illumination light beam obliquely through an objective lens having an optical axis perpendicular to the sample surface, Having a function of receiving light reflected or emitted from the same from the same direction as the irradiation direction of the irradiation light beam via the objective lens, and forming an image of the sample surface on an imaging surface of an imaging device. (Claim 1).

In this means, the sample surface is irradiated with an illumination light beam obliquely, and light reflected or emitted from the sample surface is received from the same direction as the irradiation direction of the irradiation light beam. Therefore, the ratio of the light reflected on the imaging surface of the imaging device to the specular reflection light of the irradiation light flux is reduced, so that the light is placed on a sample having a surface close to a mirror surface or an object having a high reflectance. Even when observing a sample that has been sampled, specularly reflected light is less likely to become noise, and accurate observation can be performed.

A second means for solving the above-mentioned problem is as follows.
A light source, having an optical axis perpendicular to the sample surface, condensing an illumination light beam from the light source, forming an spot on the sample surface, and light reflected or emitted from the sample surface, After condensing through the objective lens, it has a photoelectric detector for photoelectrically detecting, the optical axis of the illumination light beam,
The optical axis of the light reflected or emitted from the sample surface and condensed coincides at least on both sides of the objective lens, and the optical axis of the objective lens is at a predetermined angle (0) at the center of the objective lens. (Excluding °) and crossing each other.

In this means, the optical axis of the illuminating light beam and the optical axis of the light reflected or emitted from the sample surface and condensed coincide with each other at least on both sides of the objective lens. The optical axis of the lens forms a predetermined angle (excluding 0 °) at the center of the objective lens.
Therefore, since the specularly reflected light is reflected in a direction different from the optical axis of the received light, the proportion of the light detected by the photoelectric detector that includes the specularly reflected light of the irradiation light beam is reduced. Therefore, even when observing a sample having a surface close to a mirror surface or a sample placed on an object having high reflectivity, specular reflection light is less likely to be noise, and accurate observation is required. Can be.

The phrase "the optical axis of the illumination light beam and the optical axis of the light reflected or emitted from the sample surface and condensed at least coincide on both sides of the objective lens" means that the sample surface of the objective lens On the opposite side of the objective lens, and at least until they reach another optical element. At the beginning, the term "photoelectric detector" is a general term for those that detect light and convert the information into electrical signals.For example, photomultiplier tubes, photodiodes, phototransistors, etc. are used as photoelectric conversion elements. Are included in this. `` Photoelectric detector that photoelectrically detects after condensing through the objective lens '' is not only a photoelectric detector that directly detects light collected through the objective lens, but also light collected through the objective lens, It also includes a photoelectric detector for detecting after processing through an optical system.

[0015] A third means for solving the above problems is as follows.
A light separating means for separating the light reflected from the sample surface and the emitted light, wherein the light is emitted from the separated sample surface; Or an optical system for guiding to a photoelectric detector, wherein the optical axis of the illumination light flux and the optical axis of light that is reflected or emitted from the sample surface and collected is from the sample surface to the light separating unit. (Claim 3).

The present invention relates to a microscope, such as a fluorescence microscope, which receives light having a wavelength different from the irradiation light and performs observation. That is, it has a light separating means for separating the light reflected or emitted from the sample surface of the irradiation light from the illumination light flux, and collects the light reflected or emitted from the sample surface by the optical axis of the illumination light flux. The optical axis of the emitted light coincides from the light separating means to the sample surface. In this respect, it is similar to the conventional microscope shown in FIG.

However, in this means, the sample surface is irradiated with an illumination light beam obliquely, and light reflected or emitted from the sample surface is received through the objective lens from the same direction as the irradiation direction of the irradiation light beam. Therefore, the amount of specularly reflected light contained in the received light is reduced, and the effect as described in the first means and the second means is obtained, and only light having a wavelength different from the irradiation light is obtained. Can be observed at a higher S / N ratio.

A fourth means for solving the above problem is as follows.
The second means or the third means, wherein when the optical axis of the objective lens is the z-axis, the scanning means for scanning the spot in at least one of the x-axis direction and the y-axis direction,
The scanning means has a function of always guiding light reflected or emitted from the sample surface to a photoelectric detector even if the spot position changes simultaneously (Claim 4). .

The second means and the third means also include means for fixing the optical path and observing the sample surface by moving on the sample side. Scanning means has a function of scanning the spot and always guiding light reflected or emitted from the sample surface to the photoelectric detector even when the spot position changes. That is, since the sample surface is observed by scanning with light, higher-speed scanning can be performed with a simple mechanism than when the sample side is moved. As the scanning means used in this means, a known one as shown in the conventional microscope shown in FIG. 6 can be used.

A fifth means for solving the above problems is as follows.
The fourth means, wherein the illumination light beam applied to the objective lens is a parallel light beam, and the scanning means changes a direction of an optical axis of the illumination light, and The point at which the optical axes of the illumination light beams intersect is on the focal plane of the objective lens or a plane conjugate with the focal plane (claim 5).

In this means, the point where the optical axis of the illumination light beam intersects with the scanning means is on the focal plane of the objective lens or a plane conjugate with it. Therefore, even if the direction of the optical axis is changed by the scanning means, the optical axis of the light beam condensed on the sample surface by the objective lens always has a constant angle with respect to the optical axis of the objective lens, that is, a line perpendicular to the sample surface. Eggplant Therefore, each point on the sample surface can always be irradiated under the same conditions, and the reflected light or emitted light can always be detected under the same conditions. Since the light beam parallel to the objective lens forms a spot on the sample surface, the sample surface coincides with the focal plane of the objective lens.

A sixth means for solving the above problem is as follows.
The fifth means, wherein the scanning means is a mirror rotatable in a two-dimensional direction (Claim 6).

In this means, since a mirror which can rotate in two dimensions is used as the scanning means, it is possible to two-dimensionally scan the sample surface with one scanning means, and the structure is simple. Become.

A seventh means for solving the above-mentioned problem is as follows.
In any one of the first means to the sixth means, the optical axis of the illumination light beam and the optical axis of the objective lens may be such that specular reflection light of the illumination light beam is not detected by an imaging device or a photoelectric detector. Characterized by an angle (claim 7)
It is.

In the present means, the angle between the optical axis of the irradiation light beam and the optical axis of the objective lens is set to such an angle that such light is not detected by the imaging device or the photoelectric detector. Even when observing a sample having a surface close to a mirror surface or a sample placed on an object having a high reflectance, specular reflection light is particularly less likely to be noise,
Observation can be made accurately.

An eighth means for solving the above-mentioned problem is as follows.
In any one of the first to seventh means, the wavelength of the illumination light beam is different from the wavelength detected by the imaging device or the photoelectric detector (Claim 8).
It is.

In this means, the ratio of the reflected light of the illumination light beam entering the image pickup device or the photoelectric detector is further reduced as compared with the case where the reflected light and the fluorescent light are simply separated only by the dichroic mirror. Therefore, when used as a fluorescence microscope or the like, the noise of reflected light can be further reduced, and minute fluorescence can be observed.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram showing an optical system of a microscope according to a first embodiment of the present invention.
In FIG. 1, 1 is a laser light source, 2 is a shaping aperture, 3 is a condenser lens, 4 is a dichroic mirror, 5 is a reflecting mirror,
Reference numeral 6 denotes an objective lens, 7 denotes a sample, 8 denotes a detection lens, 9 denotes an imaging device, and 10 denotes an aperture limiting aperture.

The collimated light beam emitted from the laser light source 1 is irradiated with a converging lens 3
While being condensed, the light is reflected by the dichroic mirror 4, turned 90 °, focused on the reflecting mirror 5 and reflected. Then, the surface of the sample 7 is irradiated through the objective lens 6. In this embodiment, the focusing position of the irradiation light on the reflecting mirror 5 is determined by the focal plane of the objective lens 6.
Further, by placing the objective lens 6 at a position away from the optical axis O, the illumination light on the sample 7 is used as Koehler illumination,
In addition, it is a telecentric light beam obliquely incident.

However, in the embodiment shown in FIG. 1, the illumination light does not require any special property other than the obliquely incident property. It is preferable, but not essential, that the light reflecting position of the reflecting mirror be the focal plane of the objective lens 6.

The region of the sample 7 illuminated in this way is observed by the imaging device 9. The optical axis of the light beam observed by the imaging device 9 extends from the surface of the sample 7 to the dichroic mirror 4. It is adapted to coincide with the optical axis of the light beam. That is, the fluorescence and the reflected light emitted from the surface of the sample 7 pass through the same optical path as the irradiation light beam in the opposite direction, the opening angle is limited by the opening angle limiting aperture 10, reach the dichroic mirror 4, and the fluorescent light is reflected by the dichroic mirror 4. After passing through, the image of the surface of the sample 7 is
An image is formed on the imaging surface of the imaging device 9. That is, the surface of the sample 7 and the imaging surface of the imaging device 9 are conjugate. The imaging device 9 has an imaging surface such as a two-dimensional CCD and the like.
Can recognize a two-dimensional image of the fluorescence on the surface.

Most of the reflected light is reflected by the dichroic mirror 4 and does not reach the image pickup device 9, but a part of the light reaches the image pickup device 9 and becomes measurement noise. However, in the present embodiment, since the irradiation light beam is irradiated obliquely to the sample surface, most of the specular reflection light is reflected in a direction different from the irradiation direction and does not enter the objective lens 6. Since the light cannot pass through the aperture angle limiting aperture 10, the amount returning to the irradiation direction is small. Therefore, compared with the conventional fluorescence microscope, the amount of light received by the imaging device 9 can be further reduced, and noise can be reduced, so that an accurate fluorescent image can be observed.

When used as a normal microscope, the dichroic mirror 4 is replaced with a half mirror. Even in this case, the specular reflection light entering the imaging device 9 is very small, and only the irregular reflection light is mainly received. Therefore, when observing the surface state of a mirror-like object, accurate observation should be performed. Can be.

FIG. 2 is a schematic diagram showing an optical system of a microscope according to a second embodiment of the present invention. In the following drawings, components shown in the above-mentioned drawings except FIG. 6 are denoted by the same reference numerals, and description thereof may be omitted. FIG.
, 11 is a scan mirror, 12 and 13 are relay lenses, 14 is a photoelectric detector, and 15 is an aperture.

The parallel light beam emitted from the laser light source 1 is reflected by the dichroic mirror 4, changes its direction by 90 °, is limited in the opening angle by the opening angle limiting aperture 10, and is reflected by the scan mirror 11. Then, the light passes through the objective lens 6 and is focused on the surface of the sample 7 to form a spot.

The scan mirror 11 is rotatable about a rotation axis in a two-dimensional direction, as shown in an example to be described later, and only the position of one point of the reflection surface is not changed by the rotation. (Referred to as the center of rotation). And
The scan mirror 11 is arranged so that the optical axis of the irradiation light beam passes through the center of rotation. In the embodiment shown in FIG. 2, the scan mirror 11 is arranged so that the center of rotation of the scan mirror 11 is located on the focal plane of the objective lens 6 and away from the optical axis O of the objective lens 6. I have.

Therefore, when the scan mirror 11 rotates around the center of rotation as shown by the arrow, the optical axis of the objective lens 6 changes as shown in the figure, and the position of the spot in the x-axis direction changes. The spot is always formed on the surface of the sample 7, and the optical axis of the light beam condensed through the objective lens 6 has a direction perpendicular to the surface of the sample 7 (see FIG. (the z-axis direction) and a constant angle θ.

The reflected light and the fluorescent light from the position corresponding to the spot on the sample surface are converted into parallel light through the objective lens 6, reflected by the scan mirror 11, and the aperture angle limiting aperture 10 limits the aperture angle. And reaches the dichroic mirror 4. Among them, the fluorescent light passes through the dichroic mirror 4, is collected by the first relay lens 12, is converted into parallel light again by the second relay lens 13, and is detected by the photoelectric detector 14. . Most of the reflected light is reflected by the dichroic mirror 4, but part of the reflected light passes through the dichroic mirror 4 and is detected by the photoelectric detector 14.

An aperture 15 is provided on the focal plane of the relay lens 12, and only light passing through the aperture 15 can reach the photoelectric detector 14. And
The optical axis of the optical system formed by the relay lenses 12 and 13 and the photoelectric detector 14 passes through the center of rotation of the scan mirror 11. As a result, of the light reflected or emitted from the position corresponding to the spot on the surface of the sample 7, only the light that enters the objective lens 6 and passes through the aperture angle limiting aperture 10 enters the photoelectric detector 12. Can be. The opening angle limiting aperture 10 may be provided between the dichroic mirror 4 and the relay lens 12 or the like to limit the opening angle of only received light.

In this embodiment, as described above, the optical axis of the light focused on the surface of the sample 7 and the photoelectric detector 1
The optical axis of the light detected at 4 coincides from the dichroic mirror 4 to the sample surface 7. Therefore, most of the light specularly reflected on the surface of the sample 7 cannot pass through the objective lens 6 and the aperture limiting aperture 10, and does not reach the dichroic mirror 4. Therefore, the amount of reflected light that enters the photoelectric detector 14 can be extremely reduced, and the photoelectric detector 14 can detect fluorescence with an excellent S / N ratio.

In the above description, the scanning in the x-axis direction has been described. However, the scanning in the y-axis direction can also be performed using the scan mirror 11, and the same operation and effect as those in the scanning in the x-axis direction can be obtained. Occurs.

Note that the optical system shown in FIG. 2 can also be used as a normal microscope by replacing the dichroic mirror 4 with a half mirror. Even in this case, the specular reflection light entering the imaging device 9 is very small, and only the irregular reflection light is mainly received. Therefore, when observing the surface state of a mirror-like object, accurate observation should be performed. Can be.

It is apparent that the same operation and effect can be obtained even if the scan mirror 11 is replaced with a normal mirror and scanning is performed by moving the sample 7 mechanically instead of scanning light. .

FIG. 3 is a schematic diagram showing an optical system of a microscope according to a third embodiment of the present invention. In this embodiment,
This is an example in which scanning in the x-axis direction and scanning in the y-axis direction are performed by separate scan mirrors. In FIG. 3, 11 'is an x-axis direction scan mirror, 11 "is a y-axis direction scan mirror, 16, 1
7 is a relay lens.

The parallel light beam emitted from the laser light source 1 is reflected by the dichroic mirror 4 and changes its direction by 90 °. After the opening angle is restricted by the opening angle limiting aperture 10, the light is scanned by the y-axis direction scanning mirror 11 ″. The light is reflected while changing its axial direction, and the relay lenses 17 and 16 are reflected.
After passing through, the light becomes parallel light again, is reflected by the x-axis direction scan mirror 11 ′ while changing its direction in the x-axis direction, passes through the objective lens 6, focuses on the surface of the sample 7, and forms a spot. .

Each of the scan mirrors 11 'and 11 "is rotatable about one axis. The scan mirrors 11' and 11" are arranged so that the optical axis of the illuminating light beam passes on the rotation axis. The position of the spot is not changed by the rotation of the scan mirror 11 ″, and the position of the spot in the x-axis direction is not changed by the rotation of the y-axis scan mirror 11 ″.

In the embodiment shown in FIG. 3, a portion corresponding to the optical axis of the illumination light at the center axis of rotation of the scan mirror 11 ′ is located on the focal plane of the objective lens 6 and the optical axis O of the objective lens 6. The scan mirror 11 ′ is arranged so as to be located at a distance from the camera in the x-axis direction. And scan mirror 1
A portion corresponding to the optical axis of the illumination light at the rotation center axis of 1 ″ is located at a position conjugate with the focal plane of the objective lens 6 and away from the optical axis O of the objective lens 6 in the y-axis direction. Is provided with a scan mirror 11 ″. Both the irradiation light beam and the optical axis of the detected light pass through the rotation center axis of the scan mirror 11 ′ and the scan mirror 11 ″.

The arrangement of the other optical systems is the same as that shown in FIG. Since it is apparent to those skilled in the art that the optical system shown in FIG. 3 is equivalent to that shown in FIG. 2 and produces the same function and effect, further explanation of the function will be omitted. In this embodiment, the aperture limiting aperture 10 is also provided with the dichroic mirror 4.
And the relay lens 12 or the like, the opening angle of only the received light may be limited.

FIG. 4 shows the scan mirror 11 in FIG.
FIGS. 3A and 3B are schematic views for illustrating a method of mounting the device, wherein FIG. 3A is a plan view, FIG. 3B is a front view, and FIG. In FIG. 4, 11a is a reflection surface, 18 is a frame, 19 is an x-direction rotation axis, and 20 is a y-axis rotation axis. The frame 18 is rotated about the x-axis rotation axis, and the scan mirror 11 is rotated.
Rotates around a y-axis direction rotation shaft 20 attached to the frame 18 in a direction perpendicular to the x-axis direction rotation shaft 19. The cross section of the scan mirror 11 has a square shape as shown in (c), and the reflection surface 11a has an x-axis rotation shaft 19 and a y-axis.
It is designed to be flush with the central axis of the axial rotation shaft 20. Therefore, the intersection of the center axis of the x-axis rotation shaft 19 and the center axis of the y-axis rotation shaft 20 is the center of rotation.

The x-axis scan mirror 11 'and the y-axis scan mirror 11 "shown in FIG. 3 are obtained by removing the frame 18 and the x-axis rotation shaft 19 from those shown in FIG. In this case, a portion corresponding to the central axis of the y-axis direction rotation shaft 20 becomes the rotation center.

FIG. 5 is a diagram showing the relationship between the cross section of the irradiation light, the specular reflection light, and the detection light on the projection-side focal plane (xy plane with the optical axis O being the z axis) of the objective lens. In the figure, (a, b) is the scanning center of the irradiation light, and corresponds to the rotation center of the scan mirror 11 in FIG. A is x
It shows the area through which the illumination light passes when scanning in the direction and in the y direction. B is a region where the specularly reflected light passes through this surface, and the radius is in the same range as A around (−a, −b). C indicates an area that passes through the objective lens and the aperture limiting aperture and is detected by the photoelectric detector.

In such a relationship, the area B and the area C
Specularly reflected light is selected by selecting the scanning center (a, b) of the illumination light and the radius of the illumination light at the projection-side focal plane of the objective lens, the objective lens diameter, and the aperture angle limiting aperture diameter so as not to overlap. Can be prevented from entering the detector.

The objective lens 6 is a system that satisfies the sine condition by eliminating spherical aberration and coma. Assuming that the focal length of the objective lens 6 is f and the opening half angle of the irradiation light incident on the surface of the sample 7 is θ, the area B is substantially (x + a) ² + (y + b) ²
<(F · sin θ) ² , and the region C is substantially represented by a circle with its center at (p, q) and a radius of r, and (xp) ² + (yq) ² <r ² . Here, r is substantially represented by f · sin θ ′, where θ ′ is the half angle of the reflected light in the objective lens 6.
Assuming that the illumination light and the received light have the same optical axis, p
= A, q = b. Since f is often determined by other factors, a, b, θ, and θ ′ are determined in consideration of these relationships so that the region B and the region C do not overlap.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing an optical system of a microscope according to a first embodiment of the present invention.

FIG. 2 is a schematic diagram showing an optical system of a microscope according to a second embodiment of the present invention.

FIG. 3 is a schematic diagram showing an optical system of a microscope according to a third embodiment of the present invention.

FIG. 4 is a schematic diagram illustrating a method of attaching a scan mirror in FIG. 2;

FIG. 5 is a diagram illustrating a relationship between cross sections of irradiation light, specular reflection light, and detection light on a projection-side focal plane of an objective lens.

FIG. 6 is a schematic diagram showing an example of an optical system of a conventional confocal microscope.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Laser light source, 2 ... Molding aperture, 3 ... Condensing lens, 4
... dichroic mirror, 5 ... reflecting mirror, 6 ... objective lens, 7 ... sample, 8 ... detection lens, 9 ... imaging device, 10 ...
Aperture limiting aperture, 11 ... scan mirror, 11
a ... reflection surface, 11 '... x-axis direction scan mirror, 11 "
... Y-axis direction scan mirror, 12, 13 ... Relay lens, 14 ... Photoelectric detector, 15 ... Aperture, 16, 1
7 ... relay lens, 18 ... frame, 19 ... x direction rotation axis, 2
0 ... Y axis rotation axis

Claims (8)

[Claims] 1. A sample surface is irradiated with an illumination light beam obliquely through an objective lens having an optical axis perpendicular to the sample surface, and light reflected or emitted from the sample surface is irradiated through the objective lens. A microscope having a function of receiving light from the same direction as the irradiation direction of a light beam and forming an image of the sample surface on an imaging surface of an imaging device. 2. A light source having an optical axis perpendicular to the sample surface,
An objective lens that collects an illumination light beam from the light source to form a spot on the sample surface, and a photoelectric device that photoelectrically detects light reflected or emitted from the sample surface after condensing the light through the objective lens. An optical axis of the illumination light beam and an optical axis of the light reflected or emitted from the sample surface and condensed at least on both sides of the objective lens, and the optical axis of the objective lens is provided. Means a crossing at a predetermined angle (excluding 0 °) at the center of the objective lens. 3. The microscope according to claim 1, wherein a light separating unit that separates light reflected from the sample surface and light emitted from the sample surface, and light emitted from the separated sample surface. The optical axis of the illumination light flux, and the optical axis of the light that is reflected or emitted from the sample surface and is collected from the sample surface. A microscope, which is identical up to the light separating means. 4. The microscope according to claim 2, wherein when the optical axis of the objective lens is the z-axis, the spot is scanned in at least one of the x-axis direction and the y-axis direction. Means for scanning, said scanning means having a function of always guiding light reflected or emitted from said sample surface to a photoelectric detector even when said spot position changes. 5. The microscope according to claim 4, wherein the illumination light beam applied to the objective lens is a parallel light beam, and the scanning unit changes a direction of an optical axis of the illumination light. The microscope is characterized in that a point where the scanning means and the optical axis of the illumination light beam intersect is on a focal plane of the objective lens or a plane conjugate with the focal plane. 6. The microscope according to claim 5, wherein the scanning unit is a mirror that can rotate in a two-dimensional direction. 7. One of claims 1 to 6
The microscope according to any one of the preceding claims, wherein an optical axis of the illumination light beam and an optical axis of the objective lens are at an angle such that specular reflection light of the illumination light beam is not detected by an imaging device or a photoelectric detector. And a microscope. 8. One of claims 1 to 7
2. The microscope according to claim 1, wherein the wavelength of the illumination light beam is different from the wavelength detected by the imaging device or the photoelectric detector.