JP2003029153A - Laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope which permits selective use of both of the function of a scanning type and a total reflection type. SOLUTION: The laser microscope 2 of the scanning type has an irradiation means which irradiates the top of a specimen 38, with a beam emitted from a light source 4 passing through an objective lens 34 and generates fluorescence from this specimen 38. Furthermore, the microscope as a total reflection type microscope has, in addition to the constitution described, a lens 10 which condenses the beam to a position conjugate with the pupil position of the objective lens 34 and a parallel flat plate 12, which makes the beam incident on a position offset at a prescribed distance parallel to this beam and offset from the center of the objective lens 34, refracts the beam by the objective lens 34, makes the beam incident diagonally on the specimen 38 and makes the beam at the boundary of cover glass 36 undergo total reflection and the specimen 38 and has an inserting and removing device 8, which inserts the condenser lens 10 and the parallel plane plate 12 respectively into the irradiation means by the device, capable of inserting and removing the same and holding means which holds a laser scanner 16 at a prescribed angle.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is used in applications such as biology and medicine, and is provided with a laser scanning device.
The present invention relates to a laser microscope which irradiates a laser beam on a specimen to detect fluorescence from the excited specimen and obtains an image.

[0002]

2. Description of the Related Art A conventional scanning laser microscope of this type will be described with reference to FIG. The scanning laser microscope 102 causes a laser light source 104 to emit a laser beam (hereinafter referred to as a beam) in a predetermined direction, and a beam expander 106 expands this beam to an arbitrary diameter and emits it. Also, the dichroic mirror 108
The incident beam is reflected at a right angle by the pair of galvanometer scanners 110x and 110x.
With y, scanning is performed in the X and Y directions, respectively. Then, the beam is condensed at a predetermined distance via the pupil projection lens 112,
The light is reflected by the folding mirror 114 to the imaging lens 116, and this beam is applied to the sample 122 on which the cover glass 120 is placed via the objective lens 118.

As a result, from the sample 122, the sample 1
The fluorescence corresponding to the characteristics of 22 is emitted. This fluorescence is
The light enters the dichroic mirror 108 via the objective lens 118 and the imaging lens 116. The fluorescence that has entered the dichroic mirror 108 is transmitted and is focused by the confocal pinhole 126 via the confocal lens 124. Then, only the fluorescence selected through the laser cut filter 128 is incident on the photomultiplier tube 130, and photoelectrically converted into an electric signal according to the amount of incident light.

Then, this electric signal is stored in the image memory in synchronization with the scanning angles of the galvanometer scanners 110x and 110y, and is read out in synchronization with the video signal to display the image on the image monitor.

In such a scanning laser microscope, the use is specified and the versatility is limited. As a microscope that solves this problem, Japanese Unexamined Patent Publication No. Hei 6 (1994)
The scanning laser microscope disclosed in Japanese Patent Publication No. 27385 is known.

The scanning laser microscope 144 shown in FIG.
As shown in FIG. 7, a switchable first optical path (optical path for microscope observation: solid line) 146 and a second optical path (optical path for macroscopic observation: broken line) 148 are provided.

First, the case where the optical path of this microscope is set to the first optical path 146 for microscope observation will be described.

The microscope 144 includes a laser light source 150.
Laser beam emitted from (hereinafter referred to as beam)
Are incident on the beam expander 152. The beam expander 152 includes a focusing lens 154, a pinhole 156, a line filter 158, a collimator lens 160, and a mirror 162 in this order. In addition,
The collimator lens 160 and the mirror 162 are installed on the first slider 164 that can be integrally inserted and removed on the optical path.

Then, the beam is reflected through the mirror 166 and is incident on the dichroic mirror 168 tilted with respect to the beam. This dichroic mirror 16
8 transmits this beam and reflects it through the mirror 170. It also reflects the beam through the mirror 172. The mirror 172 is installed on the second slider 174 that can be inserted and removed on the optical path.

Then, the beam is scanned through the galvanometer scanner 176, and the beam is condensed at a predetermined distance through the pupil projection lens 178. Then, the beam is condensed and irradiated onto the sample 182 via the objective lens 180.

The fluorescence emitted from the sample 182 is traced back through the objective lens 180, the pupil projection lens 178, etc.
It is incident on the dichroic mirror 168. The dichroic mirror 168 reflects fluorescence and obtains an image of the sample 182 via the spot projection lens 184, the confocal pinhole 186, the laser cut filter 188, and the photomultiplier tube 190 in this order.

On the other hand, the case where this microscope is set to the second optical path 148 for macroscopic observation will be described. In this case, the first and second sliders 164,
174 is removed from the optical path for microscope observation.

Instead, the beam that has passed through the line filter 158 is made incident on another collimator lens 192 having a diameter larger than that of the collimator lens 160, and collimated. Then, the beam is condensed near the galvanometer scanner 176 through the collector lens 194,
Reflect this beam. Then, this beam is made incident on the observation optical prism 196 via the pupil projection lens 178, and this prism 196 transmits the beam. Then, the sample 182 is irradiated with a beam through the objective lens 180.

The fluorescence emitted from the sample 182 is made incident on the prism 196 through the objective lens 180, is reflected plural times inside the prism 196, and is emitted in a predetermined direction. This fluorescence is visually observed through the laser cut filter 198.

Therefore, by removing the observation optical prism 196 from the optical path and inserting the first and second sliders 164 and 174 into the optical path, an optical system for confocal microscope observation similar to the conventional one is constructed.

On the other hand, when the observation optical prism 196 is inserted in the optical path and the first and second sliders 164 and 174 are removed from the optical path, an optical system for visual observation is constructed.

Therefore, in the microscope having the above structure, the two sliders 164 and 174 can be inserted into and removed from the optical path and the same laser light source 150 can be used as a light source for microscope observation and a light source for macroscopic observation. it can.

By the way, in recent years, functional analysis of living cells has been actively conducted. Among these functional analysis of cells, in order to analyze only the function of the cell membrane, in order to detect only fluorescence from the cell membrane and its vicinity,
A total reflection microscope has been used.

The total internal reflection type microscope is a kind of near-field optical (laser) microscope. This microscope excites a sample with an evanescent field at the interface between the sample and the cover glass, which have different refractive indexes, and labels fluorescence corresponding to the characteristics of the sample. In this microscope, the difference in the refractive index between the cover glass and the sample (mainly the cell membrane) is used, and the illumination light (laser beam) is obliquely directed from the position offset from the center of the objective lens toward the cover glass. Lead. The laser beam is totally reflected inside the cover glass, and the sample is excited by the laser beam slightly exuding from the cover glass. The evanescent field decays exponentially with distance from the interface. In the visible light region, the interface between glass and water (live cells) is 100 nm
It is easy to obtain a bleeding depth of 200 nm, and this bleeding depth is obtained as a spatial resolution in the depth direction.

[0020]

However, the above-mentioned general scanning laser microscope and Japanese Patent Application Laid-Open No. 6-273.
The microscope disclosed in Japanese Patent Publication No. 85 may not exhibit sufficient performance, especially as an apparatus for analyzing the function of the cell membrane. Further, if both the scanning laser microscope and the total reflection laser microscope suitable for analyzing the function of the cell membrane are prepared, there is a drawback that the cost becomes high.

Further, the microscope according to Japanese Patent Laid-Open No. 6-27385 has two optical paths and two observation systems, but does not have the functions of two different microscopes.

The present invention has been made under such circumstances.
In addition to having the function as a scanning laser microscope, it also has the function as a total reflection laser microscope with a simple operation, and it is possible to provide a laser microscope that can be selectively used by switching to each microscope. To aim.

[0023]

In order to solve the above-mentioned problems, a laser microscope of the present invention comprises a laser light source, a laser scanning device for scanning a laser beam emitted from the laser light source, and an objective of the laser beam. The sample is provided with an irradiation unit that irradiates the sample through the lens to generate fluorescence from the sample, and a detection unit that detects the fluorescence so as to obtain an image of the sample. Then, a condenser lens for condensing the laser beam at a position conjugate with the pupil position of the objective lens, and a predetermined distance parallel to the laser beam are offset to a position offset from the center of the objective lens. A transparent cover material, which is arranged so as to be in contact with the sample and which has a flat surface in contact with the sample, is made to enter the laser beam and refract it with the objective lens to obliquely enter the laser beam with respect to the sample. , Or a transparent sample holding member that is held so that the sample is in contact with the sample and that has a flat surface in contact with the sample; an offset unit that totally reflects the laser beam at an interface with the sample; From the first position and the optical path, where the offset means is individually or integrally put in the optical path of the laser beam And it is characterized in that it comprises further a to a second position and allows insertion and removal of insertion and removal means.

With this structure, the scanning laser microscope can be used also as a total reflection laser microscope, and the function can be selected according to the application, so that the cost can be reduced. .

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) First, a first embodiment will be described with reference to FIGS. First, the case of performing total reflection laser microscope observation with the naked eye will be described with reference to FIG.

As shown in FIG. 1, the laser microscope 2 is
A laser light source 4 that emits a laser beam (hereinafter, referred to as a beam) in a predetermined direction from an emission port (not shown) is provided. A beam expander 6 having a plurality of optical members and expanding a beam to a predetermined diameter is provided in front of the laser light source 4. An insertion / removal device (insertion / removal means) 8 is provided in front of the beam expander 6. This insertion / removal device 8 is provided with a condenser lens 10 and a plane-parallel plate (refractive optical system) 12 in this order on the optical path of the beam. The condenser lens 10 condenses the beam substantially at the center between galvanometer scanners 16x and 16y described later.

The plane-parallel plate 12 has two planes parallel to each other and is pivotally supported about an axis perpendicular to the planes. Therefore, this plane-parallel plate 1
When the beam incident on one of the two parallel two surfaces is to be emitted from the other surface, the optical path of the beam emitted from the exit is determined according to the rotation angle of the parallel plane plate 12. The light can be emitted in parallel with a predetermined offset.

The insertion / removal device 8 can be inserted or removed from the optical path manually or electrically. That is, the insertion / removal device 8 can be manually or electrically moved to a first position arranged on the optical path of the beam and a second position removed from the optical path. In addition, these condenser lenses 10
It is also possible to insert and remove the parallel plane plate 12 as a single body from the optical path manually or electrically. That is, the condenser lens 10 and the plane parallel plate 12 can be manually or electrically moved to the first position and the second position, respectively. Here, these condenser lenses 1
0 and the plane parallel plate 12 are arranged at the first position.

In front of the insertion / removal device 8, a dichroic mirror 14 tilted by 45 ° with respect to the optical path of the beam is provided. This dichroic mirror 1
Reference numeral 4 reflects a beam and transmits light in a wavelength range longer than this beam. Therefore, the beam which is offset from the center of the mirror 14 in a predetermined direction and is incident is reflected at a right angle.

In the optical path of this beam, a pair of galvanometer scanners (laser scanning devices) 16x and 16y are provided in parallel with each other, and each is inclined at a desired angle with respect to the optical path of the beam. These scanners 16x and 16y are movably formed so as to move beams in the X direction and the Y direction, respectively, and scan a sample 38 described later. In addition, the X direction here,
The Y direction is taken on the interface between the cover glass 36 and the sample 38, which will be described later. Further, here, the galvanometer scanners 16x and 16y are used while being held in a state of being inclined at a desired angle (here, 45 °) with respect to the beam. That is, the beam reflected by the dichroic mirror 14 is converted into a pair of galvanometer scanners 16x and 16y.
The light is made incident on a position offset from the center of the in a predetermined direction. Since the beams are reflected at right angles, the beam reflected by the galvanometer scanner 16y has an optical path parallel to the beam reflected by the dichroic mirror 14.

An irradiation means for irradiating the sample 38 with the beam is provided on the optical path of this beam. The irradiation means includes a pupil projection lens 18, and the pupil projection lens 18
The beam is made incident at a position offset from the center of. Then, this beam is refracted at an arbitrary angle and emitted.

In front of the pupil projection lens 18, a turret 20 having first and second cubes 20a and 20b is arranged. A pivot is provided in the center of the first cube 20a and the second cube 20b.
Therefore, the turret 20 can be rotated around the pivot axis, and the first cube 20a and the second cube 20b can be selectively arranged with respect to the optical path.

The first cube 20a is provided with a mirror 22 tilted at a predetermined angle with respect to the optical path. A dichroic mirror 24 and a laser cut filter 26 are arranged in the second cube 20b. An opening (not shown) is provided in the direction in which the beam enters and exits the turret 20. Here, the mirror 22 provided on the first cube 20a is arranged on the optical path. That is, the beam emitted from the pupil projection lens 18 is reflected by the mirror 22.

On the optical path of this beam, the observation optical prism 2 is provided with a dichroic coating that transmits the incident beam and reflects the fluorescence from the sample.
8 are provided. The observation optical prism 28
Can be inserted into and removed from the optical path. That is, the observation optical prism 28 can be moved to a first position where it is arranged on the optical path of the beam and a second position where it is removed from the optical path. Here, the observation optical prism 28 is arranged at the first position.

An imaging lens 30 is provided on the optical path of this beam. The transmitted beam is incident on a position offset from the center of the imaging lens 30 by a predetermined distance. The beam emitted from this lens 30 is focused on a pupil position 32 of an objective lens 34 described later.

The pupil position 32 and the position approximately halfway between the galvanometer scanners 16x and 16y are:
It is set to conjugate.

On the optical path of this beam, the objective lens 34
Is provided. Generally, an objective lens having a high NA is used for total reflection laser microscope observation. This is because the observation target on which this kind of observation method is performed, that is, the sample 38 is generally a living cell, and therefore its refractive index is basically the same as that of water (1.33). Therefore, in order to cause total reflection at the interface between the cover glass 36 and the sample 38, the NA of the objective lens 34 must be larger than 1.33.

Then, the beam is incident on a position offset from the center of the objective lens 34. In front of this objective lens 34, a transparent cover material (here, a cover glass) 36 that transmits or refracts the beam is provided. The cover glass 36 has a flat surface in contact with the sample 38. When the microscope 2 is an inverted type, a sample holding member such as the bottom of a petri dish or a slide glass usually works as a transparent cover material 36.

The beam emitted from the objective lens 34 is obliquely irradiated (incident) on the cover glass 36. This beam is totally reflected at the interface between the cover glass 36 and the sample 38. Then, the sample 38 is excited by the excitation light (evanescent field) that slightly exudes from the interface of the cover glass 36, and fluorescence corresponding to the characteristics of the sample is generated.

The fluorescent light emitted from the sample 38 is observed through the objective lens 34 and the imaging lens 30 to the observation optical prism 2.
8. This fluorescence is reflected a plurality of times inside this prism 28 and emitted in a predetermined direction. A laser cut filter 40 that emits only desired fluorescence is provided on the optical path of the fluorescence. Therefore, with the naked eye, the specimen 38
Can be observed.

Next, FIG. 2 shows the case of performing total reflection laser microscope observation with an image pickup device such as a CCD camera.
Will be explained. Hereinafter, the same members will be denoted by the same reference numerals as used above, and detailed description thereof will be omitted.

The turret 20 is rotated and a dichroic mirror 24 provided on the second cube 20b is provided.
And the laser cut filter 26 are arranged on the optical path. The dichroic mirror 24 reflects a beam and transmits light in a wavelength range longer than this beam. Therefore, similarly to the case where the mirror 22 is used, the beam emitted from the pupil projection lens 18 is reflected.

The observation optical prism 28 is located at the second position and is removed from the optical path. Therefore, the beam reflected by the dichroic mirror 24 is directly incident on the imaging lens 30.

Therefore, the fluorescence according to the characteristics of the sample 38 is traced back to the objective lens 34 and the imaging lens 30 in order, and is made incident on the dichroic mirror 24 and transmitted through this mirror 24, and this fluorescence is transmitted to the laser cut filter 26. Make it incident. An imaging device (here, a CCD camera) 42 is provided on the optical path of this fluorescence. Therefore, this fluorescence can be imaged by the CCD camera 42 and displayed on a screen (not shown) for observation.

Finally, the case of observing with a scanning laser microscope will be described with reference to FIG.

The insertion / removal device 8 is at the second position and is removed from the optical path. That is, the condenser lens 10 and the plane-parallel plate 12 are at the second position and are removed from the optical path. Further, the turret 20 is rotated, and the mirror 22 provided on the first cube 20a is arranged on the optical path. Further, the observation optical prism 28 is also in the second position and is removed from the optical path.

Therefore, the beam emitted from the laser light source 4 is expanded in diameter through the beam expander 6 and is made incident on the dichroic mirror 14 substantially at the center thereof. This dichroic mirror 14 is a galvanometer scanner 1
Reflect the beam in the 6x direction. As described above, the galvanometer scanners 16x and 16y are
It is movably formed.

The beam reflected by the mirror 22 through the pupil projection lens 18 passes through the imaging lens 30 and the objective lens 3
4. The objective lens 34 forms a spot on the sample 38 with this beam. Then, the spot 38 is scanned on the sample 38 in accordance with the scanning angles of the galvanometer scanners 16x and 16y. The fluorescence generated according to the characteristics of the sample 38 is converted into the objective lens 34, the imaging lens 30,
The mirror 22, the pupil projection lens 18, and the galvanometer scanners 16y and 16x are traced back in order and made incident on the dichroic mirror 14. This dichroic mirror 14
This fluorescence is transmitted. Also, on the optical path of this fluorescence,
A confocal lens 44 is provided to collect the incident fluorescence at a predetermined focal length. A pinhole 46 is provided at this light collecting position. A laser cut filter 48 is provided on this optical path to cut a beam in a predetermined wavelength range. A photomultiplier tube 50 is provided in front of the filter 48. The beam incident on the photomultiplier tube 50 is photoelectrically converted into an electric signal according to the amount of incident light. Then, this electric signal can be accumulated in the image memory in synchronization with the scanning angles of the galvanometer scanners 16x and 16y, read out in synchronization with the video signal, and an image can be displayed on the image monitor for observation.

Therefore, with the above configuration, the scanning laser microscope observation and the total reflection laser microscope observation with the naked eye or a CCD camera can be switched and used. Therefore, one microscope can provide a microscope having two functions.

In this embodiment, the plane parallel plate 12 is used.
Is movable between the first position and the second position, but when used as a scanning laser microscope, it is possible to arrange two parallel surfaces at right angles to the optical path of the beam. It may remain in the first position.

(Second Embodiment) Next, a second embodiment will be described with reference to FIG. Since this embodiment has the same parts as those of the first embodiment, the description of these parts will be omitted. Also,
The same reference numerals are used for the members used in the first embodiment, and the description is omitted.

As shown in FIG. 4, the laser microscope 2 is
The controller 60 is provided. The controller 60 is connected to a condenser lens turret 62, a parallel plane plate driving device 64, a turret 20, an observation optical prism driving device 66, and a revolver 68 of the objective lens 34, which will be described later.

A condenser lens turret 62 is provided between the beam expander 6 and the dichroic mirror 14. The condensing lens turret 62 includes a condensing lens 62a, a condensing lens system 62b having a longer focal length than that of the single lens condensing lens 62a by a combination of a convex lens and a concave lens, and a hole (not shown). There is. That is, the condenser lens turret 62 has three stages. When this hole is arranged on the optical path of the beam emitted from the beam expander 6, the beam is allowed to go straight while maintaining the state of the collimated light. The condenser lens turret 62 is controlled by the controller 60.

Therefore, when the scanning laser microscope observation is performed, the condenser lens turret 62 has a hole set on the optical path. Further, when performing total reflection laser microscope observation, the condenser lens 62a or the condenser lens system 62b is arranged on the optical path. Then, the condenser lens 62a and the condenser lens system 62b will be described.

As described above, generally, an objective lens having a high NA is used for observation with a total reflection laser microscope. For example, when the NA of the objective lens is 1.65, there is a margin in the refractive index difference (1.65-1.33 = 0.22), so that the beam emitted from the imaging lens 30 toward the objective lens 34 is NA can be increased. However, when the NA of the objective lens 34 is 1.4, the refractive index difference is only 0.07, and for efficient illumination, the NA of the beam emitted from the imaging lens 30 to the objective lens 34 is used.
Needs to be fairly small. Further, the NA of the outgoing beam from the imaging lens 30 to the objective lens 34 is
If the value is too small, the illumination range cannot be sufficiently obtained.

When the luminous flux of the beam emitted from the beam expander 6 is Φo, the luminous flux Φob of the beam emitted from the objective lens 34 can be expressed by the following equation.

Φob = Φo × F ′ / (F × B) where B is the magnification of the objective lens 34, F ′ is the focal length of the pupil projection lens 18, and F is the focusing lens 62a and the focusing lens system 62b. The focal length.

Therefore, when performing total reflection laser microscope observation, the NA of the beam emitted from the imaging lens 30 to the objective lens 34 takes into consideration the difference between the NA of the objective lens 34 and the refractive index of the sample 38. It is necessary to set the optimum value.

Therefore, in the present embodiment, the condenser lens 62a depends on the type of the objective lens 34 set in the optical path.
Alternatively, the condenser lens system 62b is selected and installed. The objective lens 34 is attached to the revolver 68, and the controller 60 controls the objective lens 34 installed on the optical path.
The type is automatically determined, and the condenser lens turret 62 is driven.

Next, the plane parallel plate 12 will be described.
The plane-parallel plate 12 is installed on the plane-parallel plate driving device 64. The plane-parallel plate 12 is arranged between the condenser lens turret 62 and the dichroic mirror 14. The plane-parallel plate 12 is rotatably supported by a plane-parallel plate driving device 64 so as to be rotatable in a plane having a beam optical path.

The rotation angle of the plane parallel plate 12 is set depending on whether the scanning laser microscope observation is performed or the total reflection laser microscope observation is performed. In addition, when the total reflection laser microscope observation is performed, it is set according to the type of the objective lens 34 set in the optical path. The objective lens 34 is attached to the revolver 68, and the controller 60 controls the objective lens 34 installed on the optical path.
Is detected, and the plane-parallel plate 12 is set to the rotation angle corresponding to this. That is, the optimum offset amount of the beam is set depending on the type of the objective lens 34 set in the optical path. This value differs depending on both the magnification of the objective lens 34 and the NA.

When the scanning laser microscope observation is performed, the plane parallel plate 1 is irrespective of the type of the objective lens 34.
2 is set perpendicular to the optical path of the beam.

Next, the control system of the microscope 2 will be described. When performing the scanning laser microscope observation, the condenser lens turret 62 is set as a hole. The plane-parallel plate 12 is set perpendicular to the optical path by the plane-parallel plate drive device 64. Further, a mirror 22 is controlled to be set on the turret 20, and the observation optical prism 2
8 is controlled by the observation optical prism driving device 66 so as to be removed from the optical path.

When the total reflection laser microscope observation is performed, the turret 20 is selected depending on whether the macroscopic observation or the CCD camera 42 is used.
The mirror 22 is controlled so as to be installed on the optical path when performing the macroscopic observation, and the dichroic mirror 24 and the laser cut filter 26 are respectively installed at the optical paths when performing the observation by the CCD camera 42.

Further, depending on the objective lens 34 used,
The angle of the condenser lens turret 62 and the plane parallel plate 12 is set.

In the present embodiment, the condenser lens 62a and the condenser lens system 62b are arranged in the condenser lens turret 62, but instead of these, a condenser lens having a zoom function is used to change the focal length. You may Alternatively, a plurality of condenser lenses having different focal lengths may be arranged in the condenser lens turret 62 and these condenser lenses may be switched and used.

Therefore, in this embodiment, the controller 60 can be used to electrically switch between the scanning laser microscope observation and the total reflection laser microscope observation. Also,
When performing total reflection laser microscope observation, optimal illumination can be performed on the objective lens 34 used.

In the second embodiment, when the scanning laser microscope observation is performed, the plane parallel plate 12 is controlled so as to be at right angles to the beam, but like the first embodiment. Alternatively, it may be moved to the second position.

In the first and second embodiments,
As the means for offsetting the beam, the parallel plane plate (refractive optical system) 12 has been described, but if the beam can be offset such as by using a plurality of mirrors (reflection optical system), another optical system is used. It doesn't matter.

The parallel plane plate 12 is provided between the laser light source 4 and the dichroic mirror 14, but, for example, the galvanometer scanner 16y and the pupil projection lens 1 are provided.
It may be provided at another position, such as between 8 and the like.

Further, in the first embodiment, the irradiation means is shown to point from the pupil projection lens 18 to the objective lens 34, but the beam expander 6 to the pupil projection lens 18 are also included.

In the above-mentioned microscope, the erecting type scanning microscope has been described as an example, but the present invention is not limited to this.
For example, an inverted microscope may be used.

Although some embodiments have been specifically described with reference to the drawings, the present invention is not limited to the above-described embodiments, and is carried out without departing from the scope of the invention. Includes all implementations.

[0075]

As described above, according to the present invention,
It is possible to provide a laser microscope which has a function as a scanning laser microscope and also has a function as a total reflection laser microscope by a simple operation, and which can selectively use each microscope. Therefore, the cost can be significantly reduced as compared with the case where both the scanning type and the total reflection type laser microscopes are prepared.

[Brief description of drawings]

FIG. 1 is a schematic explanatory view of a laser microscope according to a first embodiment when performing total internal reflection laser microscope observation with the naked eye.

FIG. 2 is a schematic explanatory view of a laser microscope when performing total reflection laser microscope observation with the image pickup apparatus according to the first embodiment.

FIG. 3 is a schematic explanatory diagram of a laser microscope according to the first embodiment when performing confocal scanning laser microscope observation.

FIG. 4 is a schematic explanatory view showing a laser microscope according to a second embodiment.

FIG. 5 is a schematic explanatory view showing a scanning laser microscope according to a conventional embodiment.

FIG. 6 is a schematic explanatory view showing a scanning laser microscope according to a conventional embodiment.

[Explanation of symbols]

2 ... Laser microscope, 4 ... Laser light source, 8 ... Inserting / removing device, 10 ... Condensing lens, 12 ... Parallel plane plate, 16x, 1
6y ... Galvanometer scanner, 34 ... Objective lens, 3
6 ... Cover material, 38 ... Specimen

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G043 AA03 BA16 CA05 EA01 GA02                       GA03 GB01 GB03 HA01 HA02                       HA09 JA02 KA02 KA09 LA02                       LA03                 2H052 AA00 AA07 AA08 AA09 AB05                       AB14 AB24 AB25 AC04 AC09                       AC14 AC15 AC22 AC24 AC27                       AC34 AD32 AF14

Claims (3)

[Claims] 1. A laser light source, a laser scanning device for scanning a laser beam emitted from the laser light source, and an irradiation for irradiating a specimen with the laser beam through an objective lens to generate fluorescence from the specimen. Means for detecting the fluorescence so as to obtain an image of the sample, and a condenser lens for condensing the laser beam at a position conjugate with the pupil position of the objective lens, The laser beam is offset by a predetermined distance in parallel with the laser beam, and the laser beam is made incident on a position offset from the center of the objective lens, and is refracted by the objective lens so that the laser beam is oblique to the sample. A cover material that is incident and is arranged so as to be in contact with the sample and that is transparent and has a flat surface in contact with the sample, Alternatively, a transparent sample holding member held so that the sample is in contact with the sample and having a flat surface in contact with the sample, offset means for totally reflecting the laser beam at an interface with the sample, the condenser lens and the offset And a means for inserting and removing the means into a first position and a second position to be removed from the optical path of the laser beam, respectively. Laser microscope. 2. The laser microscope according to claim 1, wherein the offset means includes a refraction optical system that refracts a laser beam or a reflection optical system that reflects the laser beam. 3. The condensing lens comprises a zoom mechanism capable of changing its focal length, or a plurality of condensing lenses each having a different focal length, which are switchable. The laser microscope described.