JP2012003284A - Total reflection type microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope which permits selective use of both functions of a scanning type and a total reflection type.SOLUTION: A scanning type laser microscope 2 has irradiation means which irradiates the top of a sample 38 with a beam emitted from a light source 4 through an objective lens 34 and generates fluorescence from the sample 38. Furthermore, the microscope as a total reflection type microscope includes, in addition to the above-described constitution, a lens 10 which condenses the beam to a position conjugate with a pupil position of the objective lens 34, and a parallel flat plate 12, which makes the beam incident on a position offset at a predetermined distance parallel to the beam and offset from the center of the objective lens 34, makes the beam be refracted by the objective lens 34 to make the beam incident obliquely on the sample 38, and makes the beam total reflect at a boundary between a cover glass 36 and the sample 38, and has an inserting/removing device 8 which inserts each of the condenser lens 10 and the parallel flat plate 12 into the irradiation means by the device capable of inserting/removing the same, and holding means which holds a laser scanning device 16 at a predetermined angle.

Description

  The present invention relates to a laser microscope that is used in applications such as biology and medicine, and that has a laser scanning device and obtains an image by detecting fluorescence from a sample excited by irradiating the sample with a laser beam.

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

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

  The electric signal is stored in the image memory in synchronization with the scanning angle of the galvanometer scanners 110x and 110y, read out in synchronization with the video signal, and displayed on the image monitor.

Such a scanning laser microscope has a problem that its application is specified and versatility is limited. As a microscope that solves this problem, a scanning laser microscope disclosed in JP-A-6-27385 is known.
As shown in FIG. 6, the scanning laser microscope 144 includes a switchable first optical path (microscope observation optical path: solid line) 146 and a second optical path (visual observation optical path: broken line) 148. ing.

First, the case where the optical path of this microscope is set to the first optical path 146 for microscopic observation will be described.
The microscope 144 causes a laser beam (hereinafter referred to as a beam) emitted from the laser light source 150 to enter 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 order. The collimator lens 160 and the mirror 162 are installed on a first slider 164 that can be inserted and removed integrally 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. The dichroic mirror 168 transmits the beam and reflects the beam through the mirror 170. Further, the beam is reflected through the mirror 172. The mirror 172 is installed on a second slider 174 that can be inserted and removed on the optical path.
Then, the beam is scanned through the galvanometer scanner 176, and this beam is condensed at a predetermined distance through the pupil projection lens 178. Then, the beam is condensed and irradiated on the specimen 182 through the objective lens 180.

  Fluorescence emitted from the specimen 182 is incident on the dichroic mirror 168 in the order of the objective lens 180, the pupil projection lens 178, and the like. The dichroic mirror 168 reflects fluorescence and obtains an image of the sample 182 through 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 visual observation will be described. In this case, the first and second sliders 164 and 174 are removed from the optical path for microscope observation from the above configuration.
Instead, the beam that has passed through the line filter 158 is incident on another collimator lens 192 having a diameter larger than that of the collimator lens 160 to be parallel light. Then, the beam is condensed near the galvanometer scanner 176 via the collector lens 194, and this beam is reflected. The beam is incident on the observation optical prism 196 via the pupil projection lens 178, and the prism 196 transmits the beam. Then, the sample 182 is irradiated with a beam through the objective lens 180.
Fluorescence emitted from the specimen 182 is incident on the prism 196 through the objective lens 180, is reflected a plurality of times within the prism 196, and is emitted in a predetermined direction. This fluorescence is visually observed through a laser cut filter 198.

  Therefore, when the observation optical prism 196 is removed from the optical path and the first and second sliders 164 and 174 are placed on the optical path, an optical system for confocal microscope observation similar to the conventional one is configured.

On the other hand, when the observation optical prism 196 is placed on 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 configured.
Therefore, in the microscope having the above-described configuration, the two sliders 164 and 174 can be inserted 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 visual observation.

By the way, in recent years, functional analysis of living cells has been actively performed. Among these functional analyzes of cells, a total reflection microscope has been used to detect only the fluorescence from the cell membrane and its vicinity, particularly in order to analyze the function of the cell membrane.
The total reflection microscope is a kind of near-field optical (laser) microscope. In this microscope, the specimen is excited by an evanescent field at the interface between the specimen and the cover glass having different refractive indexes, and fluorescence corresponding to the characteristics of the specimen is labeled. In this microscope, using the difference in refractive index between the cover glass and the specimen (mainly cell membrane), illumination light (laser beam) is obliquely directed toward the cover glass from a position offset from the center of the objective lens. Lead. The laser beam is totally reflected in the cover glass, and the specimen is excited by the laser beam that slightly oozes from the cover glass. The evanescent field decays exponentially as it moves away from the interface. In the visible light region, it is easy to obtain a bleeding depth of 100 nm to 200 nm at the interface between glass and water (live cells), and this bleeding depth is obtained as a spatial resolution in the depth direction.

However, the above-described general scanning laser microscope and the microscope disclosed in Japanese Patent Laid-Open No. 6-27385 may not exhibit sufficient performance particularly 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 disadvantage that the cost increases.
Further, in the microscope disclosed in Japanese Patent Laid-Open No. 6-27385, two optical paths are provided and two observation systems are provided. However, the microscope does not have a function as two different microscopes.

  The present invention is made under such circumstances, and has a function as a scanning laser microscope, and also has a function as a total reflection laser microscope with a simple operation, and can be selectively used by switching to each microscope. It is an object of the present invention to provide a laser microscope that can be used.

In order to solve the above problems, a laser microscope according to the present invention irradiates a laser light source, a laser scanning device that scans a laser beam emitted from the laser light source, and a sample through the objective lens. And an irradiating means for generating fluorescence from the specimen, and a detecting means for detecting the fluorescence so as to obtain an image of the specimen. Then, a condensing lens for condensing the laser beam at a position conjugate with the pupil position of the objective lens, and a predetermined distance offset parallel to the laser beam, and a position offset from the center of the objective lens A cover material that is incident on the laser beam and is refracted by the objective lens so that the laser beam is obliquely incident on the sample and is disposed so as to contact the sample and has a flat surface in contact with the sample. Or a transparent specimen holding member held so that the specimen is in contact with it and a flat surface in contact with the specimen, offset means for totally reflecting the laser beam at the interface with the specimen, the condenser lens, and the From the optical path and a first position where the offset means is individually or integrally placed on 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.
By adopting such a configuration, the scanning laser microscope can be used as a total reflection laser microscope, and the function can be selected according to the application, so that the cost can be reduced.

  According to the present invention, there is provided a laser microscope that has a function as a scanning laser microscope and also has a function as a total reflection laser microscope with a simple operation and can selectively use each microscope. Can be provided. For this reason, it is possible to greatly reduce the cost compared to arranging both the scanning and total reflection laser microscopes.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic explanatory diagram of a laser microscope in a case where total reflection laser microscope observation is performed with the naked eye according to the first embodiment. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic explanatory diagram of a laser microscope in the case of performing total reflection laser microscope observation with an imaging apparatus according to the first embodiment. The schematic explanatory drawing of the laser microscope in the case of performing confocal scanning laser microscope observation concerning 1st Embodiment. Schematic explanatory drawing which shows the laser microscope concerning 2nd Embodiment. Schematic explanatory drawing which shows the scanning laser microscope concerning the conventional embodiment. Schematic explanatory drawing which shows the scanning laser microscope concerning the conventional embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
First, a first embodiment will be described with reference to FIGS. 1 to 3.
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 includes 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). A beam expander 6 having a plurality of optical members and expanding the 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. The insertion / removal device 8 includes a condensing lens 10 and a plane-parallel plate (refractive optical system) 12 in order on the beam optical path. The condensing lens 10 condenses the beam substantially at the center between galvanometer scanners 16x and 16y described later.
The parallel flat plate 12 has two surfaces parallel to each other, and is pivotally supported so as to be rotatable around an axis perpendicular to these surfaces. For this reason, when the beam incident on one of the two parallel surfaces of the plane-parallel plate 12 is emitted from the other surface, the beam is emitted from the exit according to the rotation angle of the plane-parallel plate 12. The beam can be emitted with a predetermined distance offset parallel to the optical path of the beam.

  The insertion / removal device 8 can be inserted / removed manually or electrically from the optical path. In other words, the insertion / removal device 8 can be moved manually or electrically between a first position where it is disposed on the optical path of the beam and a second position where it is removed from the optical path. Further, the condenser lens 10 and the plane-parallel plate 12 can be inserted and removed manually or electrically from the optical path as a single unit. That is, the condenser lens 10 and the plane parallel plate 12 can be moved manually or electrically to the first position and the second position, respectively. Here, the condensing lens 10 and the plane parallel plate 12 are disposed at the first position.

A dichroic mirror 14 that is inclined 45 ° with respect to the optical path of the beam is provided in front of the insertion / removal device 8. The dichroic mirror 14 reflects a beam and transmits light in a wavelength region longer than the beam. For this reason, the incident beam offset in a predetermined direction from the center of the mirror 14 is reflected at a right angle.
A pair of galvanometer scanners (laser scanning devices) 16x and 16y are provided in parallel to each other in the beam optical path, and are inclined at a desired angle with respect to the beam optical path. These scanners 16x and 16y are formed so as to be movable so as to scan a specimen 38 to be described later by moving the beam in the X direction and the Y direction, respectively. Here, the X direction and the Y direction are taken on an interface between a cover glass 36 and a specimen 38, which will be described later. Further, here, the galvanometer scanners 16x and 16y are used while being held at a desired angle (45 ° in this case) with respect to the beam. That is, the beam reflected by the dichroic mirror 14 is incident on a position offset in a predetermined direction from the center of the pair of galvanometer scanners 16x and 16y. Since these beams are reflected at right angles, the beam reflected by the galvanometer scanner 16 y has an optical path parallel to the beam reflected by the dichroic mirror 14.

Irradiation means for irradiating the specimen 38 with the beam is provided on the beam optical path. This irradiating means includes a pupil projection lens 18 and makes a beam incident at a position offset from the center of the pupil projection lens 18. Then, the beam is refracted at an arbitrary angle and emitted.
In front of the pupil projection lens 18, a turret 20 including first and second cubes 20a and 20b is disposed. A pivot is provided at the center between the first cube 20a and the second cube 20b. For this reason, 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 that is inclined at a predetermined angle with respect to the optical path. The second cube 20b is provided with a dichroic mirror 24 and a laser cut filter 26. An opening (not shown) is provided in the direction in which the beam enters and exits the turret 20. Here, the mirror 22 provided in 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, there is provided an observation optical prism 28 with a dichroic coating that transmits the incident beam and reflects the fluorescence from the specimen. The observation optical prism 28 can be inserted / removed from / on the optical path. That is, the observation optical prism 28 can be moved to a first position where it is disposed on the optical path of the beam and to a second position where it is removed from the optical path. Here, the observation optical prism 28 is disposed 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 by a predetermined distance from the center of the imaging lens 30. The beam emitted from the lens 30 is condensed at a pupil position 32 of an objective lens 34 to be described later.
The pupil position 32 and the substantially intermediate position between the galvanometer scanners 16x and 16y are set conjugate.

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

Then, the beam is made incident at a position offset from the center of the objective lens 34. In front of the 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 specimen 38. When the microscope 2 is an inverted type, a sample holding member such as a petri dish bottom or a slide glass usually serves as a transparent cover member 36.
The beam emitted from the objective lens 34 is irradiated (incident) onto the cover glass 36 obliquely. This beam is totally reflected at the interface between the cover glass 36 and the specimen 38. Then, the specimen 38 is excited by excitation light (evanescent field) that slightly oozes from the interface of the cover glass 36, and fluorescence corresponding to the characteristics of the specimen is generated.

  Fluorescence emitted from the specimen 38 is incident on the observation optical prism 28 via the objective lens 34 and the imaging lens 30. The fluorescence is reflected a plurality of times within the prism 28 and emitted in a predetermined direction. A laser cut filter 40 for emitting only desired fluorescence is provided on the fluorescence optical path. Therefore, the specimen 38 can be observed with the naked eye.

  Next, the case of performing total reflection laser microscope observation with an imaging device such as a CCD camera will be described with reference to FIG. Hereinafter, the same symbols are used for the same members, and detailed descriptions thereof are omitted.

The turret 20 is rotated, and a dichroic mirror 24 and a laser cut filter 26 provided on the second cube 20b are arranged on the optical path. The dichroic mirror 24 reflects a beam and transmits light in a wavelength region longer than the beam. For this reason, the beam emitted from the pupil projection lens 18 is reflected as in the case of using the mirror 22.
The observation optical prism 28 is at the second position and is removed from the optical path. For this reason, the beam reflected by the dichroic mirror 24 is directly incident on the imaging lens 30.
Accordingly, the fluorescence corresponding to the characteristics of the specimen 38 goes back in order through the objective lens 34 and the imaging lens 30 and enters the dichroic mirror 24 and is transmitted through the mirror 24 so that the fluorescence is incident on the laser cut filter 26. An imaging device (in this case, a CCD camera) 42 is provided on the fluorescent light path. Therefore, this fluorescence can be imaged by the CCD camera 42 and displayed on a screen (not shown) for observation.

Finally, the case of observation with a scanning laser microscope will be described with reference to FIG.
The insertion / removal device 8 is in the second position and is removed from the optical path. That is, the condensing lens 10 and the plane-parallel plate 12 are in the second position and are removed from the optical path. Further, the turret 20 is rotated, and a mirror 22 provided on the first cube 20a is disposed on the optical path. The observation optical prism 28 is also in the second position and is removed from the optical path.

  Accordingly, the diameter of the beam emitted from the laser light source 4 is enlarged via the beam expander 6 and is incident on the approximate center of the dichroic mirror 14. The dichroic mirror 14 reflects the beam toward the galvanometer scanner 16x. As described above, the galvanometer scanners 16x and 16y are formed to be movable.

The beam reflected by the mirror 22 via the pupil projection lens 18 is incident on the objective lens 34 via the imaging lens 30. The objective lens 34 forms a spot on the specimen 38 with this beam. Then, the sample 38 is scanned with spots according to the scanning angle of the galvanometer scanners 16x and 16y. The fluorescence generated in accordance with the characteristics of the specimen 38 is incident on the dichroic mirror 14 in the order of the objective lens 34, the imaging lens 30, the mirror 22, the pupil projection lens 18, and the galvanometer scanners 16y and 16x. The dichroic mirror 14 transmits this fluorescence. In addition, a confocal lens 44 is provided on the fluorescent light path, and the incident fluorescent light is condensed at a predetermined focal length. A pinhole 46 is provided at this condensing 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 in accordance with the amount of incident light. This electric signal can be stored in the image memory in synchronization with the scanning angle of the galvanometer scanners 16x and 16y, read out in synchronization with the video signal, and displayed on the image monitor for observation.
Therefore, with the above-described configuration, the scanning laser microscope observation and the total reflection laser microscope observation with the naked eye or the CCD camera can be switched and used. For this reason, a microscope having two functions can be provided with one microscope.

  In this embodiment, the plane-parallel plate 12 is movable between the first position and the second position. However, when used as a scanning laser microscope, two parallel planes are used as the beam optical path. If it can be arranged at a right angle to it, it may remain in the first position.

(Second Embodiment)
Next, a second embodiment will be described with reference to FIG. Since the present embodiment includes the same parts as the configuration of the first embodiment, description of these parts will be omitted. Moreover, about the member used in 1st Embodiment, description is abbreviate | omitted using the same code | symbol.

As shown in FIG. 4, the laser microscope 2 includes a controller 60. 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 condensing lens turret 62 is provided between the beam expander 6 and the dichroic mirror 14. The condenser lens turret 62 includes a condenser lens system 62b having a focal length longer than that of a single lens condenser lens 62a by a combination of a condenser lens 62a, a convex lens and a concave lens, and a hole (not shown). Yes. That is, the condenser lens turret 62 is switched in three stages. When this hole is arranged on the optical path of the beam emitted from the beam expander 6, this beam is made to go straight while maintaining the state of the collimated light. The condenser lens turret 62 is controlled by the controller 60.

  Therefore, when performing observation with a scanning laser microscope, the condensing lens turret 62 has a hole on the optical path. When performing total reflection laser microscope observation, the condenser lens 62a or the condenser lens system 62b is disposed on the optical path. 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 total reflection laser microscope observation. 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 The 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 in order to perform efficient illumination, the NA of the outgoing beam from the imaging lens 30 to the objective lens 34 is considerably large. It needs to be small. Further, the NA of the outgoing beam from the imaging lens 30 to the objective lens 34 is not necessarily small, and if this value is too small, a sufficient illumination range cannot be obtained.

If the light beam emitted from the beam expander 6 is Φo, the light beam Φob emitted from the objective lens 34 is expressed by the following equation.
Φob = Φo × F ′ / (F × B)
Here, B is the magnification of the objective lens 34, F ′ is the focal length of the pupil projection lens 18, and F is the focal length of the condenser lens 62a and the condenser lens system 62b.
Therefore, when the total reflection laser microscope observation is performed, the NA of the outgoing beam from the imaging lens 30 to the objective lens 34 is an optimum value in consideration of the difference between the NA of the objective lens 34 and the refractive index of the specimen 38. It is necessary to make it.

  Therefore, in the present embodiment, the condenser lens 62a or the condenser lens system 62b is selected and installed depending on 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 automatically determines the type of the objective lens 34 installed on the optical path and drives the condenser lens turret 62.

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

The rotation angle of the parallel flat plate 12 is set for each of the cases of observation with a scanning laser microscope and observation with a total reflection laser microscope. In the case of performing total reflection laser microscope observation, the setting is made according to the type of objective lens 34 set in the optical path. The objective lens 34 is mounted on the revolver 68, and the controller 60 detects the type of the objective lens 34 installed on the optical path, and the parallel plane plate 12 is set at the corresponding rotation angle. That is, the optimum offset amount of the beam is set according to the type of the objective lens 34 set in the optical path. This value varies depending on both the magnification of the objective lens 34 and the NA.
When performing observation with a scanning laser microscope, the plane-parallel plate 12 is set perpendicular to the beam optical path regardless of the type of the objective lens 34.

  Next, the control system of the microscope 2 will be described. When performing observation with a scanning laser microscope, the condensing lens turret 62 is set to a hole. The plane parallel plate 12 is set perpendicular to the optical path by the plane parallel plate driving device 64. The turret 20 is controlled so that the mirror 22 is set, and the observation optical prism 28 is controlled by the observation optical prism driving device 66 so as to be removed from the optical path.

  When performing the total reflection laser microscope observation, the turret 20 is selected depending on whether the observation is performed with the naked eye or the CCD camera 42. When performing macroscopic observation, the mirror 22 is controlled so that the dichroic mirror 24 and the laser cut filter 26 are respectively installed on the optical path when the CCD camera 42 is used for observation.

Further, the condenser lens turret 62 and the angle of the plane parallel plate 12 are set by the objective lens 34 to be used.
In this embodiment, the condenser lens turret 62 is provided with the condenser lens 62a and the condenser lens system 62b, but instead of these, a condenser lens having a zoom function is used, and the focal length is variable. Good. Alternatively, a plurality of condensing lenses having different focal lengths may be arranged in the condensing lens turret 62, and these may be switched and used.

  Therefore, in this embodiment, the controller 60 can be used to electrically switch between scanning laser microscope observation and total reflection laser microscope observation. In addition, when performing observation with a total reflection laser microscope, the objective lens 34 to be used can be optimally illuminated.

In the second embodiment, when performing observation with a scanning laser microscope, the plane-parallel plate 12 is controlled so as to be perpendicular to the beam. However, as in the first embodiment, the second plane is the same as the second embodiment. You may make it move to this position.
In the first and second embodiments, the plane-parallel plate (refractive optical system) 12 has been described as means for offsetting the beam. However, the beam may be changed by using a plurality of mirrors (reflective optical system). Other optical systems may be used as long as they can be offset.

The plane parallel plate 12 is provided between the laser light source 4 and the dichroic mirror 14, but may be provided at other positions, for example, between the galvanometer scanner 16y and the pupil projection lens 18.
In the first embodiment, the irradiation unit is shown to point from the pupil projection lens 18 to the objective lens 34. However, the irradiation unit includes the beam expander 6 to the pupil projection lens 18.
In the above-described microscope, an erecting scanning microscope has been described as an example. However, the present invention is not limited to this, and for example, an inverted microscope may be used.

  Although several embodiments have been specifically described so far with reference to the drawings, the present invention is not limited to the above-described embodiments, and all the embodiments performed without departing from the scope of the invention are not limited thereto. Including implementation.

  DESCRIPTION OF SYMBOLS 2 ... Laser microscope, 4 ... Laser light source, 8 ... Insertion / removal apparatus, 10 ... Condensing lens, 12 ... Parallel plane plate, 16x, 16y ... Galvanometer scanner, 34 ... Objective lens, 36 ... Cover material, 38 ... Sample.

The present invention relates to a total reflection microscope.

An object of the present invention is to provide a total reflection microscope in which an optimum offset amount of light is set according to the type of objective lens set in the optical path .

In order to solve the above problems, the total reflection type microscope according to the present invention includes a light source, is irradiated on the specimen through the objective lens selected light from plural kinds from the light source, the fluorescence from the specimen irradiating means for generating a detection means for detecting the fluorescence to obtain an image of the specimen, the light collector for focusing the light provided on the light path of the light at the pupil position conjugate with the position of the objective lens a lens, incident light from the light source position is offset with respect to the center of the objective lens selected from the plurality of kinds, is incident the light obliquely to the specimen is refracted by the objective lens A transparent cover material disposed so as to be in contact with the specimen and having a flat surface in contact with the specimen; or a transparent specimen holding member in which the specimen is in contact with the specimen and a specimen holding member having a flat surface in contact with the specimen; With the specimen And offset means for totally reflecting the light in the plane, and detects the type of the objective lens selected from the plurality of types, the condensing position of the pupil position conjugate with the position of the objective lens, are selected from the plurality of kinds And a control unit for controlling the offset means to an offset amount in accordance with the type of objective lens .

ADVANTAGE OF THE INVENTION According to this invention , the total reflection type microscope by which the optimal offset amount of light is set with the kind of objective lens set to the optical path can be provided .

Claims (3)

A laser light source;
A laser scanning device that scans a laser beam emitted from the laser light source;
Irradiation means for irradiating the laser beam on the specimen through the objective lens and generating fluorescence from the specimen;
Detecting means for detecting the fluorescence so as to obtain an image of the specimen;
In a laser microscope equipped with
A condensing lens for condensing the laser beam at a position conjugate with the pupil position of the objective lens;
The laser beam is incident on a position offset by a predetermined distance in parallel to the laser beam and offset from the center of the objective lens, and is refracted by the objective lens so that the laser beam is inclined with respect to the specimen. A transparent cover material arranged so as to be in contact with the specimen and having a flat surface in contact with the specimen, or a transparent specimen holding member having a flat face in contact with the specimen, which is held in contact with the specimen. Offset means for totally reflecting the laser beam at the interface with the specimen;
Insertion / removal means that can be inserted / removed into / from a first position where the condenser lens and the offset means are individually or integrally placed on the optical path of the laser beam and a second position where they are removed from the optical path;
A laser microscope characterized by further comprising:   The laser microscope according to claim 1, wherein the offset unit includes a refractive optical system that refracts a laser beam or a reflective optical system that reflects the laser beam.   3. The laser microscope according to claim 1, wherein the condensing lens includes a zoom mechanism that can change a focal length thereof, or a plurality of condensing lenses that have different focal lengths.

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