JP2003177325A - Total reflection fluorescence microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a total fluorescence microscope which can realize stable fluorescent observation by total reflection illumination and can improve operability. <P>SOLUTION: The total reflection fluorescence microscope makes the change over of vertical illumination and total reflection illumination possible by changing the incident angle of the illumination light to be radiated to a sample through an objective lens 7 from a laser beam source, in which the incident angle of the illumination light from the objective lens 7 on the sample is regulated to a range making the total reflection illumination obtainable. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a total reflection fluorescence microscope capable of fluorescence observation by total reflection illumination.

[0002]

2. Description of the Related Art Recently, functional analysis of living cells has been actively carried out. In the functional analysis of these cells, in particular, in order to observe the function of the cell membrane, the cell membrane and its vicinity are examined. A total reflection fluorescence microscope, which acquires a total reflection fluorescence image, has been attracting attention.

In such a total reflection fluorescence microscope, total reflection illumination is used to locally illuminate only a sample near the glass surface. This total internal reflection illumination uses evanescent light that bleeds to the sample side by several hundred nm at the interface between the glass and the sample. Since background noise (scattered light, etc.) is extremely low, fluorescence of one molecule of fluorescent dye is used. Observation is possible.

By the way, in the fluorescence observation by such total reflection illumination, the exudation depth of the evanescent light exuding from the glass surface to the sample side is different due to the refractive index of the glass, and the exudation depth is However, it depends on the purpose of the spectroscope.

Therefore, conventionally, it has been considered that the incident angle of the illumination light from the glass to the sample is varied depending on the condition of the sample and the depth to be observed.

Japanese Unexamined Patent Publication (Kokai) No. 9-159922 employs such an idea. A mirror for reflecting light from a light source to the objective lens side is moved to shift the illumination light from the optical axis. The incident angle to the sample is continuously changed to switch between epi-illumination and total reflection illumination.

Further, in general, a micrometer or the like is used for the movement of the mirror that reflects the illumination light, that is, the adjustment of the incident angle from the glass to the sample because fine adjustment is necessary.

[0008]

However, in the case of Japanese Patent Laid-Open No. 9-159922, for example, when performing fluorescence observation with total internal reflection illumination, switching to epi-illumination fluorescence illumination is performed when adjusting the incident angle from the glass to the sample. In this case, it is necessary to move the mirror again to the position of total internal reflection illumination, but during this time, the sample on the glass surface is irradiated with intense epi-illumination as excitation light, There is a risk of fading the whole.

If a micrometer or the like is used to move the mirror in the range of the epi-illumination and the total reflection illumination, the micro-meter has a small movement amount per rotation of the rotary operation unit, so that the epi-illumination can be changed. When the total reflection illumination is switched, the number of rotations increases, and it takes time and effort to switch the illumination, and the operability of fluorescence observation is significantly reduced. Further, this means that if the epifluorescent illumination is switched to the total reflection illumination while the illumination excitation light is being applied to the sample, the entire sample may be discolored during this switching.

The present invention has been made in view of the above circumstances, and provides a total reflection fluorescence microscope capable of realizing stable fluorescence observation by total reflection illumination and near total reflection illumination and improving operability. The purpose is to

[0011]

The invention according to claim 1 is
In a total internal reflection fluorescence microscope capable of switching between total reflection illumination and near total reflection illumination by changing the incident angle of illumination light emitted from a light source through an objective lens, through the objective lens of the illumination light. It is characterized in that a regulation means is provided for regulating the incident angle to the sample within a range where total reflection illumination and near total reflection illumination can be obtained.

According to a second aspect of the invention, in the first aspect of the invention, the light source has an optical fiber whose output end is movable in a direction perpendicular to the optical axis, and the regulating means is The moving range in the direction perpendicular to the optical axis of the emission end of the optical fiber is restricted to a range in which the total internal reflection illumination and near total internal reflection illumination are obtained.

According to a third aspect of the present invention, in the first aspect of the invention, the optical path between the light source and the objective lens has a reflecting member movably provided along the optical path direction, and the regulation is performed. The means limits the moving range of the reflecting member along the optical path direction to a range in which the total internal reflection illumination and the near total internal reflection illumination are obtained.

The invention according to claim 2 is the invention according to claim 1 or 2.
In the invention described above, an optical element for diffusing the illumination light is detachably provided in an optical path between the light source and the objective lens.

As a result, according to the present invention, the angle of incidence of the illumination light on the sample through the objective lens can be always regulated within the range in which total reflection illumination and near total reflection illumination can be obtained. It is possible to prevent the entire sample from being discolored by the strong light due to, and it is possible to obtain stable fluorescence observation by total reflection illumination and near total reflection illumination. Also, always
Since the incident angle of the illumination light from the objective lens to the sample can be adjusted within the range of total internal reflection illumination and near total internal reflection illumination, operability can be improved.

[0016]

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

First, the outline of total reflection fluorescence observation according to the present invention will be described with reference to FIG. In this case, an example of an inverted microscope for observing with the objective lens 7 arranged below the sample 2 is shown.

The sample 2 is provided with a cover glass 3 on the lower side thereof, and an objective lens 7 is provided below the cover glass 3 with an oil 5 interposed therebetween.

A rotatable mirror unit turret 9 holding two or more fluorescent mirror units 10a and 10b is arranged on the optical axis 8 of the objective lens 7, and the mirror unit turret 9 rotates about a rotation axis 14. The fluorescent mirror units 10a and 10b corresponding to total reflection illumination or epi-illumination are selectively switched on the optical axis 8 by operation. In the drawing, the fluorescent mirror unit 10a corresponding to the total reflection illumination is switched on the optical axis 8. A high-reflecting mirror 24 is arranged in the incident optical path of the fluorescent mirror units 10a and 10b. This high reflection mirror 24
Are fixed to the mirror holding portion 22 by adhesion or the like. The mirror holding portion 22 is provided with a dovetail portion 22a. The dovetail portion 22a is movably held in the dovetail groove portion 20 provided in the epi-illumination tube 17, and the high-reflecting mirror 24 is moved in the paper sheet vertical direction by moving the operation knob 23 in and out. It can be done. In this case, when it is on the optical axis 19 of the epi-illumination projection tube 17 as shown in the figure, the light from the laser light source 41 is reflected toward the fluorescent mirror units 10a, 10b.

On the other hand, the laser light from the laser light source 41 is
After being introduced from the optical fiber incidence part 40, it is emitted from the optical fiber emission part 38. The emitted light 21a from the optical fiber emitting portion 38 is converted into the parallel light 21b by the collimator lens 29 of the fiber light projecting tube 28, and the high reflection mirror 2
After being reflected by 4, the light is condensed by the condenser lens 18 and guided to the fluorescence mirror unit 10a. The fluorescence mirror unit 10a is provided with a dichroic mirror 11a and an absorption filter 12a, and the light condensed by the condenser lens 18 is reflected by the dichroic mirror 11a and focused on the rear focal position 6 of the objective lens 7. Connect the objective lens 7
Light emitted from the tip of the sample enters the sample 2 from the cover glass 3. Here, the light is emitted from the tip of the objective lens 7, and the cover glass (high refractive index side) 3 to the sample (low refractive index side) 2
The optical axis 31 of the emitted light 21a from the optical fiber emitting section 38 is shifted in the direction perpendicular to the optical axis 30 of the fiber light projecting tube 28 so that the incident angle of the incident light incident on the optical axis becomes larger than the critical angle. By doing so, the evanescent light 4 exuding in the range of about several hundred nm from the boundary surface with the cover glass 3 can be generated on the sample (low refractive index side) 2.

The fluorescent substance in the sample 2 existing near the surface of the cover glass 3 where the evanescent light 4 is generated is
After being excited by the evanescent light 4 that is excitation light, it emits fluorescence, passes through the objective lens 7 and the dichroic mirror 11a, and the absorption filter 12a removes the disadvantageous light in the wavelength region other than fluorescence, and then observation imaging The sample 2 is guided to the system 15 and imaged on a high-sensitivity camera (CCD etc.) 16.
The fluorescent substance inside can be observed.

On the other hand, in the case of a normal epi-illumination, as shown in FIG.
As shown in, the high-reflecting mirror 24 is removed from the optical axis 19 of the epi-illumination projection tube 17, and the fluorescence mirror unit 10a is switched to the fluorescence mirror unit 10b for epi-illumination. In this case, the fluorescence mirror unit 10b includes the dichroic mirror 11b, the absorption filter 12b and the excitation filter 13.
b of the rays from the mercury burner 26 of the mercury lamp house 25, only the excitation light is transmitted through the excitation filter 13b and reflected by the dichroic mirror 11b.
Light emitted from the tip of the objective lens 7 enters the sample 2 from the cover glass 3. Then, the fluorescence from the fluorescent substance in the sample 2 is transmitted through the dichroic mirror 11b, and the absorption filter 12b removes the detrimental light in the wavelength region other than the fluorescence, and then is guided to the observation imaging system 15. An image is formed on the high-sensitivity camera (CCD or the like) 16, and the fluorescent substance in the sample 2 can be observed.

Next, a concrete embodiment will be described.

(First Embodiment) FIGS. 3A and 3B
(C) has shown the schematic structure of the total reflection fluorescence microscope to which the 1st Embodiment of this invention was applied. In addition, in FIG.
The same parts as those in FIG. 1 are designated by the same reference numerals.

In this case, the epi-illumination tube 17 is fixed to the inverted microscope body (not shown). This epi-illuminator 1
Reference numeral 7 denotes a connecting portion 17a with a fiber light projecting tube 28 and a connecting portion 1 with a mercury lamp house 25 holding a mercury burner 26.
7b. The optical axis 30 of the fiber projection tube 28 is
The optical axis 27 of the mercury lamp house 25 is connected so as to be orthogonal to the optical axis 19 of the epi-illumination tube 17 so as to coincide with the optical axis 19 of the epi-illumination tube.

A high reflection mirror 2 is provided inside the epi-illumination tube 17.
4 reflects the parallel light 21b of the fiber illuminator 28 onto the illuminator 1
Mirror holding section 22 so that it is reflected on the optical axis 19 of 7.
It is fixed by adhesion or the like. The mirror holding portion 22 is provided with a dovetail portion 22a. The dovetail portion 22a is movably held in a dovetail groove portion 20 provided in the epi-illumination tube 17 so that the operation knob 23 can be taken in and out from the outside of the epi-illumination tube 17 in the vertical direction. Thus, the high reflection mirror 24 can be moved in the direction perpendicular to the paper surface. In addition, the mirror holder 22
Is provided with a light shielding portion 22b on the side surface on the mercury lamp house 25 side.

The fiber light projecting tube 28 comprises a collimating lens 29 and a fiber introducing section 32. A fiber introducing portion 32 is connected to an end portion of the fiber light projecting tube 28 on the side opposite to the incident light projecting tube 17 side.

On the other hand, the light emitted from the laser light source 41 enters the optical fiber 39 from the optical fiber incident section 40,
The emitted light 21a is emitted from the optical fiber emitting portion 38.
The optical fiber emitting section 38 is fixed to the moving section 37 with screws or the like (not shown). Outside surface portion 37a of the moving portion 37
Is fitted to the inner side surface portion 32a1 of the fiber introducing portion 32 and is movable in the left-right direction 54 of the drawing.

Here, the fiber introducing portion 32 is provided with a screw hole 32a having a center line 36 and a fitting hole 32b parallel to the left-right direction 54 of the paper surface of the moving portion 37, and the screw hole 32a.
A lid cylinder 33 having a threaded portion is engaged with the fitting hole 32.
An adapter 35 is fitted in b.

A compression coil spring 34, which is an elastic body, is provided between the lid cylinder 33 and the adapter 35, and the compression coil spring 34 is longer than the natural length.
It is sandwiched in a short state, and the adapter 35 is attached to the moving part 3
The outer side surface portion 37a of No. 7 is abutted. On the other hand, a slope contact portion 37b is provided on the opposite side of the outer surface portion 37a of the moving portion 37.

A micrometer holding portion 42 is fixed to the fiber introducing portion 32 with a screw or the like (not shown). A micrometer main body 44 is held on the micrometer holding portion 42 with screws or the like (not shown).

The micrometer body 44 includes a rotating portion 44a.
The knob 46 is engaged with a screw or the like (not shown). Further, in the fiber introducing portion 32, a screw hole 32a,
A cylindrical hole 32 having a center line orthogonal to the center line of the fitting hole 32b
c is provided, and the capsule adapter 43 is arranged in a state of being fitted into the cylindrical hole 32c and being sandwiched between the slope contact portion 37b and the tip end portion 44b of the rotating portion 44a of the micrometer main body 44. There is.

As a result, the knob 46 of the micrometer main body 44 is rotated, and the slope contact portion 3 of the moving portion 37 is moved by the capsule adapter 43 of the tip portion 44b of the rotating portion 44a.
7b is pressed and the moving part 37 is moved against the pressing force of the compression coil spring 34, so that the light is emitted from the tip of the objective lens 7 and the sample 3 (low refractive index side) from the cover glass (high refractive index side) 3. The optical axis 31 of the outgoing light 21a from the optical fiber emitting section 38 can be shifted (to the left in the drawing) from the optical axis 30 of the fiber light projecting tube 28 so that the incident angle of the incident light entering 2 can be adjusted. I have to.

The fixing portion 44c of the micrometer body 44 is provided with an opening 45a as a regulating means as shown in FIG.
The opening 45a of the notch stopper 45 having the U-shaped notch 45c is fitted. A screw 47 is provided to adjust the distance between the U-shaped notches 45c.

Next, the operation of the first embodiment thus constructed will be described.

In this case, the adapter 35 is in a state of pressing the outer side surface portion 37a of the moving portion 37 by the compression coil spring 34, while rotating the knob 46 of the micrometer main body 44 so that the tip end portion is rotated. 44b can be moved in the vertical direction 55 of the drawing, and the capsule adapter 4
The moving unit 37 can be moved in the left-right direction 54 of the drawing sheet via the unit 3. As a result, the optical axis 31 of the emitted light 21 a from the optical fiber emitting unit 38 is parallel to the optical axis 30 of the fiber light projecting tube 28, and the fiber light projecting tube 28 is
The adjustment can be made in the vertical direction with respect to the optical axis 30 of.

At the same time, it becomes possible to adjust the incident light angle emitted from the tip of the objective lens 7 from the cover glass 3 to the sample 2. Here, the incident angle from the cover glass 3 to the sample 2 is adjusted. The knob 46 is rotated to move the moving portion 37 so that the angle is slightly larger than the critical angle.
Adjust the position of the notch stopper 45 at that position.
The side portion 45d of the rotary portion 44 of the micrometer body 44
The notch stopper 45 is abutted against the abutment portion 44aa of a, the gap between the notch portions 45c of the notch stopper 45 is tightened with a screw 47, and the fixing portion 44c of the micrometer main body 44 is sandwiched by the opening 45a. The micrometer body 44 is positioned and fixed.

Therefore, after this, the moving portion 37 is regulated by the notch stopper 45, and the optical fiber emitting portion 38.
Cannot be moved to the optical axis 30 side of the fiber projection tube 28, and the movement is restricted only within the range of total reflection illumination in which the incident angle from the cover glass 3 to the sample 2 is larger than the critical angle.

On the other hand, a light ray (not shown) from the mercury lamp house 25 is shielded by the light shielding portion 22b of the mirror holding portion 22, but the operation knob 23 is pulled out to the front side in the direction perpendicular to the plane of the drawing, and the mirror holding portion is pulled out. By removing 22 from the optical path, a light beam (not shown) from the mercury burner 26 can be guided to the epi-illumination tube 17. At this time, the mirror unit turret 9 is rotated around the rotation shaft 14 to thereby generate the excitation filter 13b and the dichroic mirror 11b.
By arranging the fluorescence mirror unit 10b provided with the absorption filter 12b on the optical axis, normal epi-illumination illumination observation can be performed.

Therefore, according to the first embodiment, when the incident angle from the cover glass 3 to the sample 2 is adjusted by the notch stopper 45 fixed to the micrometer body, this incident angle is adjusted. Is regulated within a range larger than the critical angle, so that fluorescence observation can be performed only by total internal reflection illumination. Therefore, it is possible to prevent the entire sample from being discolored by the strong light of the epi-illumination, and it is possible to obtain stable fluorescence observation by the total-reflection illumination. Also,
Since the incident angle from the cover glass 3 to the sample 2 can always be adjusted within the range of total internal reflection illumination, operability can be improved. Furthermore, since switching between total reflection illumination and epi-fluorescence illumination can be performed quickly by inserting and removing the high-reflection mirror 24,
It is also possible to avoid the cause of fading of the sample when switching from epi-fluorescence illumination to total internal reflection illumination while illuminating the sample with illumination light (or vice versa).

Although the inverted microscope has been described in the first embodiment, the same effect can be obtained when the inverted microscope is applied.

(Modification of First Embodiment) Next, a modification of the first embodiment will be described. 4 (a) and 4 (b) explain a modification of the first embodiment, and the same parts as those in FIG. 3 are denoted by the same reference numerals.

In this case, the fiber projection tube 28 is provided with a slide opening 28a on the rear side of the collimator lens 29 (on the side of the epi-illumination projection tube 17).
A slider 48 having an opening 48a and an opening 48b is provided to be movable in the left-right direction 54 of the drawing. The slider 48 has a diffusion plate 49 fixed to the opening 48a by a ring screw (not shown).

On the other hand, the mirror holder 2 of the high-reflection mirror 24
2 is directly fixed to the fixing portion 50 of the epi-illumination projection tube 17 with a screw or the like (not shown).

With this structure, the opening 48b of the slider 48 is aligned with the optical axis 30 of the fiber projection tube 28 in the state where the total reflection illumination is set as described in the first embodiment. When it is arranged, this state is maintained as it is, but when the slider 48 is moved to arrange the opening 48a having the diffusion plate 49 on the optical axis 30, the parallel light 2 is emitted.
1b is diffused and switched to epi-fluorescent illumination.

In this way, in addition to the effects similar to those of the above-described first embodiment being selected, switching between total reflection illumination and epi-illumination is realized without using the mercury lamp house 25. be able to.

In the modification of the first embodiment, if the fiber light projection tube 28 is provided coaxially with the epi-illumination projection tube 17, the high reflection mirror 24 can be omitted and the cost is further reduced. Can have a different structure.

(Second Embodiment) Next, a second embodiment of the present invention will be described. FIG. 5 illustrates a modification of the first embodiment, and the same parts as those in FIG. 3 are designated by the same reference numerals.

In this case, the mirror holding portion 22 of the high reflection mirror 24 is provided with a dovetail portion 51.
Is held in a dovetail groove 52 provided in the epi-illumination tube 17 so as to be movable in the left-right direction 54 of the drawing.

In this case, the mirror holding portion 22 is always provided with the compression coil spring 34 via the adapter 35 and the dovetail portion 5.
The first contact portion 51a is in a pressed state. on the other hand,
A micrometer holding portion 53 is provided on a side surface of the epi-illumination tube 17 opposite to the position where the adapter 35 is arranged, and the micrometer holding portion 53 is provided with a micrometer body 44. In this case, in the micrometer body 44, the tip end portion 44b of the rotating portion 44a rotated by the rotation of the knob 46 is brought into contact with the contact portion 51b of the dovetail portion 51.

In this state, by rotating the knob 46 of the micrometer body 44, the mirror holding portion 22 is moved along the reflection optical path of the high reflection mirror 24 by the dovetail portion 51, and the parallel light at the high reflection mirror 24 is moved. The reflection position of 21b can be adjusted.

As a result, the reflected light 21c of the parallel light 21b on the high reflection mirror 24 can be shifted from the optical axis 19, and is emitted from the objective lens 7 to adjust the incident light angle from the cover glass 3 to the sample 2. can do.

Also here, the position of the mirror holding portion 22 is adjusted by rotating the knob 46 so that the incident angle from the cover glass 3 to the sample 2 is slightly larger than the critical angle, and at that position, The notch stopper 45 is brought into contact with the rotating portion 44a of the micrometer main body 44, and the notch stopper 45 is tightened to position and fix the micrometer main body 44.

As a result, after this, the mirror holding unit 2
In No. 2, the reflection light 21c from the high reflection mirror 24 cannot be moved to the optical axis 19 side due to the restriction of the notch stopper 45, and the incident angle from the cover glass 3 to the sample 2 is larger than the critical angle. It is restricted to move only within the lighting range.

In this case, similarly to the modification of the first embodiment, the slider 48 is provided and the opening 4 having the diffusion plate 49 is provided.
By arranging 8a on the optical axis 30 to diffuse the parallel light 21b, it is possible to obtain epi-illumination using the laser light source 41.

In the second embodiment, the mirror holding portion 22 is moved along the reflected light path of the high reflection mirror 24, but it may be moved along the incident light path of the high reflection mirror 24. Similar effects can be obtained.

In the above-described embodiment, the micrometer main body 44 and the notch stopper 45 for restricting the movement of the rotating portion 44a of the micrometer main body 44 are used. Light tube 28
A light-shielding plate having a slit hole that is shifted to the left or right from the center of the optical axis 30 is disposed in front of the optical fiber emitting section 38, and the optical axis of the emitted light emitted from the optical fiber emitting section 38 is projected onto the fiber. Even if it is moved to the optical axis 30 side of the tube 28,
By blocking the light on the optical axis center side of the emitted light by the light shielding plate, the switching to the epi-illumination can be prevented, and the movement can be restricted only within the range of the total reflection illumination. In this case, the switching between the total internal reflection illumination and the epi-illumination fluorescent illumination using the laser light source 41 is performed by moving the high-reflection mirror 24 so as to be inserted into and removed from the optical path, as in the first embodiment. You can

Next, a method for realizing total reflection illumination will be described. When the incident angle of the illumination light from the cover glass 3 to the sample 2 exceeds the critical angle, the confirmation method is a lens barrel (not shown).
The eyepiece (not shown) attached to the CT is changed to a CT (centering telescope) (not shown), and this CT is used to make a lens group (not shown) near the rear focal position 6 of the objective lens 7. I see. The illumination light passes through a lens group (not shown) of the objective lens 7 to cause autofluorescence of the lens group, and in particular, a bright spot can be observed in the lens group in the vicinity of the rear focus position 6 where light is focused.

When the incident angle from the cover glass 3 to the sample 2 is smaller than the critical angle, only one bright spot on the incident angle side of the illumination light is confirmed at the rear focal position 6 of the objective lens 7, but it is critical. If the angle is larger than the angle, the bright spots are symmetrically arranged about the optical axis in the vicinity of the inner side of the outer periphery of the rear focal position 6 of the objective lens 7.
One is observed. The second bright spot is because the illumination light totally reflected at the boundary surface between the cover glass 3 and the sample 2 returns to the inside of the objective lens 7 from the tip of the objective lens 7.

When the incident angle of the illumination light from the cover glass 3 to the sample 2 is changed from a state smaller than the critical angle to a state larger than the critical angle, one bright point is gradually moved from the center side of the optical axis of the objective lens toward the outer circumference. When it moves and the incident angle exceeds the critical angle, a second bright point appears symmetrically with respect to the optical axis center of the objective lens, and when this second bright point appears, the notch stopper 4
5 is fixed to the micrometer main body 44.

Next, the definition of near total reflection illumination and the operation and effect when applied to the first embodiment will be described based on the first embodiment. The configuration is similar to that of the first embodiment, and will not be described. Also, regarding the operation and effect, the same parts as those in the first embodiment will be omitted, and only different parts will be described.

First, near total reflection illumination is defined as follows. Cover glass 3 for total internal reflection lighting
To the sample 2 side, which is the low refractive index medium side at the interface between the sample 2 and
Although the evanescent light 4 is generated in the range of several hundreds nm, if the critical angle of the incident angle from the cover glass 3 to the sample 2 is made slightly smaller, the refracted light from the cover glass 3 to the sample 2 will be The light is emitted from 3 along the vicinity of the boundary surface of the sample 2. With this illumination method, it is possible to illuminate a range of several μm in the vicinity of the cover glass 3 of the sample 2. This is a kind of dark-field illumination, and in the present invention, this illumination method is called near total reflection illumination.

Next, the operation of near total reflection illumination will be described. Here, only the near total reflection illumination by the laser light source 41 is described, and the description about the epi-fluorescence illumination using the mercury burner 26 as the light source is omitted. First, similarly to the first embodiment, the knob 46 of the micrometer 44 is rotated to make total reflection illumination in which the incident angle from the cover glass 3 to the sample 2 is larger than the critical angle. Next, the knob 46 of the micrometer body 44 is gradually rotated in the direction in which the incident angle from the cover glass 3 to the sample 2 becomes smaller,
The illumination light is adjusted to the above-mentioned near total reflection illumination.

In this state, as in the first embodiment,
By positioning and fixing the notch stopper 45 with respect to the micrometer body 44, the movement of the optical fiber emitting part 38 is restricted only to the range of near total reflection illumination and total reflection illumination.

Next, the effect will be described. Even in the case of near total reflection illumination, the illumination range is only the sample 2 in the range of several μm in the vicinity of the upper surface of the cover glass 3, and it is possible to prevent discoloration of the entire sample 2 as in the first embodiment, and to illuminate the illumination light. It is possible to move away from the axis and quickly change to total internal reflection illumination.

In addition to the effects described above, it is possible to observe a region that cannot be observed by the evanescent light 4 generated by total reflection illumination, and a sample 2 that cannot be observed because the generated fluorescence is too weak. Further, in the case of epi-fluorescence illumination using the mercury burner 26 as a light source, fluorescence is emitted from the entire sample 2, whereas in near-total reflection illumination, the illumination range can be limited to a range of several μm in the vicinity of the cover glass 3, which is unnecessary. Fluorescence can be removed, and observation with less background noise becomes possible.

In addition, the present invention is not limited to the above-described embodiment, and can be variously modified at the stage of carrying out the invention without departing from the spirit of the invention.

Furthermore, the above-described embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, the problem described in the section of the problem to be solved by the invention can be solved, and it is described in the section of the effect of the invention. In the case where the effect described above is obtained, a configuration in which this constituent element is deleted can be extracted as an invention.

[0069]

As described above, according to the present invention, it is possible to provide a total reflection fluorescence microscope capable of realizing stable fluorescence observation by total reflection illumination and near total reflection illumination and improving operability. .

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram illustrating an outline of a total reflection fluorescence microscope according to the present invention.

FIG. 2 is a schematic configuration diagram for explaining normal fluorescence epi-illumination of the total internal reflection fluorescence microscope according to the present invention.

FIG. 3 is a diagram showing a schematic configuration of a first embodiment of the present invention.

FIG. 4 is a diagram showing a schematic configuration of a modified example of the first embodiment of the present invention.

FIG. 5 is a diagram showing a schematic configuration of a second embodiment of the present invention.

[Explanation of symbols]

2 ... Sample 3 ... Cover glass 4 ... Evanescent light 5 ... oil 6 ... Rear focus position 7 ... Objective lens 8 ... Optical axis 9 ... Mirror unit turret 10a. 10b ... Fluorescent mirror unit 11a, 11b ... Dichroic mirror 12a, 12b ... Absorption filter 13b ... Excitation filter 14 ... Rotation axis 15 ... Observation imaging system 16 ... High sensitivity camera 17… Epi-illumination tube 17a ... Connection part 17b ... connecting part 18 ... Condensing lens 19 ... Optical axis 20 ... Ant groove 21a ... Outgoing light 21b ... Parallel light 21c ... Reflected light 22 ... Mirror holder 22a ... Ant section 22b ... Shading section 23 ... Operation knob 24 ... High-reflection mirror 25 ... Mercury lamp house 26 ... Mercury burner 27 ... Optical axis 28 ... Fiber floodlight tube 28a ... slide opening 29 ... Collimating lens 30, 31 ... Optical axis 32 ... Fiber introduction section 32a1 ... Inner side surface 32a ... screw hole 32b ... Mating hole 32c ... Cylinder hole 33 ... Lid cylinder 34 ... Compression coil spring 35 ... Adapter 36 ... Center line 37 ... Moving part 37a ... Outer surface portion 37b ... Slope contact part 38 ... Optical fiber emitting section 39 ... Optical fiber 40 ... Optical fiber entrance 41 ... Laser light source 42 ... Micrometer holder 43 ... Capsule adapter 44 ... Micrometer body 44a ... Rotating part 44b ... Tip 44c ... Fixed part 44aa ... Abutting part 45 ... Notch stopper 45a ... opening 45c ... Notch 45d ... Side part 46 ... Knob 47 ... Screw 48 ... slider 48a, 48b ... Opening 49 ... Diffusion plate 50 ... Fixed part 51 ... Ant section 51a, 51b ... Abutting portion 52 ... Ant groove 53 ... Micrometer holder 54 ... Left and right direction on paper 55 ... Vertical direction on paper

Claims (4)

[Claims] 1. A total reflection fluorescence microscope capable of switching between total reflection illumination and near total reflection illumination by changing an incident angle of illumination light emitted from a light source through an objective lens to the sample. A total reflection fluorescence microscope, characterized in that a total reflection fluorescence microscope is provided which restricts an incident angle to the sample through an objective lens within a range where total reflection illumination and near total reflection illumination can be obtained. 2. The light source has an optical fiber whose output end is movable in a direction perpendicular to the optical axis, and the restriction means is perpendicular to the optical axis of the output end of the optical fiber. The total reflection fluorescence microscope according to claim 1, wherein a moving range in a direction is restricted to a range where the total reflection illumination and the near total reflection illumination are obtained. 3. The optical path between the light source and the objective lens is
A reflecting member provided so as to be movable along the optical path direction, wherein the regulating means sets a moving range of the reflecting member along the optical path direction to a range where the total internal reflection illumination and the near total internal reflection illumination are obtained. The total reflection fluorescence microscope according to claim 1, which is regulated. 4. The total reflection fluorescence microscope according to claim 2, wherein an optical element for diffusing the illumination light is removably provided in an optical path between the light source and the objective lens.