JP3995458B2 - Total reflection fluorescence microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a total reflection fluorescence microscope that enables fluorescence observation by total reflection illumination.
[0002]
[Prior art]
Recently, functional analysis of living cells has been actively performed. In the functional analysis of these cells, in order to observe the function of the cell membrane in particular, a total reflection fluorescence image from the cell membrane and its vicinity is used. The total reflection fluorescence microscope to be acquired has been attracting attention.
[0003]
Such a total reflection fluorescent microscope uses total reflection illumination that locally illuminates only a sample near the glass surface. This total reflection illumination uses evanescent light that oozes out to the sample side at the interface between glass and sample, and the background noise (scattered light, etc.) is extremely low. Observation is possible.
[0004]
By the way, in such fluorescence observation with total reflection illumination, the depth of evanescent light that oozes out from the glass surface to the sample side varies depending on the refractive index of the glass, etc. This is whether you want to observe to a certain depth, depending on the purpose of the speculum.
[0005]
Therefore, conventionally, it has been considered to change the incident angle of illumination light from the glass to the sample depending on the condition of the specimen and the depth to be observed.
[0006]
Japanese Patent Application Laid-Open No. 9-159922 adopts such an idea, and moves a mirror that reflects light from a light source toward the objective lens, and shifts illumination light from the optical axis to shift from the glass to the sample. The incident angle is continuously changed to switch between epi-illumination and total reflection illumination.
[0007]
In general, the movement of the mirror that reflects the illumination light, that is, the adjustment of the incident angle from the glass to the sample requires a fine adjustment, and therefore a micrometer or the like has been used.
[0008]
[Problems to be solved by the invention]
However, in the thing of Unexamined-Japanese-Patent No. 9-159922, when performing fluorescence observation with total reflection illumination, for example, it may switch to epifluorescence illumination at the time of adjustment of the incident angle from glass to a sample. It is necessary to move the mirror to the position of total reflection illumination again, but during this time, the sample on the glass surface is irradiated with strong epi-illumination as excitation light, and the entire sample may fade. .
[0009]
If a micrometer or the like is used to move the mirror in the range from epi-illumination to total reflection illumination, the micrometer has a small amount of movement per rotation of the rotation operation unit, so that the epi-illumination to total reflection illumination When switching, the number of rotations increases, and it takes time to switch, and the operability of fluorescence observation is significantly reduced. In addition, this means that if switching from epi-fluorescence illumination to total reflection illumination with illumination excitation light applied to the sample, the entire sample may be faded during this switching.
[0010]
The present invention has been made in view of the above circumstances, and an object thereof is to provide a total reflection fluorescent microscope capable of realizing stable fluorescence observation by total reflection illumination and near total reflection illumination and improving operability. And
[0011]
[Means for Solving the Problems]
  The invention according to claim 1 is a light source.And illumination opticsThrough the objective lens,Changing the incident angle of the illumination light irradiated to the sampleA moving unit included in the illumination optical system, and moving the moving unit.In a total reflection fluorescent microscope that enables switching between total reflection illumination and near total reflection illumination,
  A fine movement mechanism for finely moving the moving section, an operation section for operating the fine movement mechanism, and a regulating means for regulating a movement range of the moving section,
The moving range isA range in which the angle of incidence of the illumination light on the sample through the objective lens can provide total reflection illumination and near total reflection illuminationIsIt is characterized by that.
[0012]
  The invention according to claim 2 is the invention according to claim 1,The illumination optical system includes:Having an optical fiber,The moving unit is configured to connect the output end of the optical fiber.Provided to be movable in the direction perpendicular to the optical axisAndThe regulating means isBy operation of the operation unitThe moving range in the direction perpendicular to the optical axis of the output end of the optical fiber is restricted to a range in which the total reflection illumination and near total reflection illumination can be obtained.
[0013]
  The invention according to claim 3 is the invention according to claim 1,The illumination optical system includes:Having a reflective member,The moving unit moves the moving member along the incident optical path of illumination light to the reflecting member, or along the optical axis direction of the reflected light path,The regulating means is theOptical axis of incident optical path or reflected optical pathThe moving range along the direction is restricted to a range in which the total reflection illumination and near total reflection illumination can be obtained.
[0014]
  Claim4The invention as described in claim 1 or3In the described invention,Illumination opticsAn optical element for diffusing the illumination light is detachably provided in the optical path.
[0015]
As a result, according to the present invention, the incident angle of the illumination light to the sample can always be regulated within a range where total reflection illumination and near total reflection illumination can be obtained. Thus, fading of the entire sample can be prevented, and stable fluorescence observation by total reflection illumination and near total reflection illumination can be obtained. In addition, since the incident angle of the illumination light from the objective lens to the sample can always be adjusted in the range of total reflection illumination and near total reflection illumination, operability can be improved.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0017]
First, an overview 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 in which observation is performed using the objective lens 7 disposed below the sample 2 is shown.
[0018]
In the sample 2, a cover glass 3 is disposed on the lower side, and an objective lens 7 is provided below the cover glass 3 with oil 5 interposed therebetween.
[0019]
A rotatable mirror unit turret 9 holding two or more fluorescent mirror units 10a and 10b is disposed on the optical axis 8 of the objective lens 7, and all of the mirror unit turret 9 is rotated around the rotation axis 14 by rotating operation. The fluorescent mirror units 10a and 10b corresponding to reflected illumination or epi-illumination are selectively switched on the optical axis 8. In the drawing, the fluorescent mirror unit 10 a corresponding to the total reflection illumination is switched on the optical axis 8. A high reflection mirror 24 is disposed in the incident optical path of the fluorescent mirror units 10a and 10b. The high reflection mirror 24 is fixed to the mirror holding portion 22 by bonding or the like. The mirror holding part 22 is provided with an ant part 22a. The dovetail part 22a is held in the dovetail groove part 20 provided in the incident light projection tube 17 so as to be movable in the direction perpendicular to the paper surface. By moving the operation knob 23 in and out, the highly reflective mirror 24 is moved in the direction perpendicular to the paper surface. It can be made to. In this case, when it is on the optical axis 19 of the epi-illumination projection tube 17 as shown in the drawing, the light from the laser light source 41 is reflected toward the fluorescent mirror units 10a and 10b.
[0020]
On the other hand, the laser light from the laser light source 41 is introduced from the optical fiber entrance 40 and then emitted from the optical fiber exit 38. The emitted light 21a from the optical fiber emitting section 38 is converted into parallel light 21b by the collimating lens 29 of the fiber projection tube 28, reflected by the high reflection mirror 24, collected by the condenser lens 18, and fluorescent. It is guided to the mirror unit 10a. The fluorescent mirror unit 10 a is provided with a dichroic mirror 11 a and an absorption filter 12 a, and the light collected by the condenser lens 18 is reflected by the dichroic mirror 11 a and is focused on the rear focal position 6 of the objective lens 7. The light emitted from the tip of the objective lens 7 enters the sample 2 from the cover glass 3. Here, an optical fiber is used so that the incident angle of incident light that is emitted from the tip of the objective lens 7 and enters the sample (low refractive index side) 2 from the cover glass (high refractive index side) 3 becomes larger than the critical angle. If the optical axis 31 of the emitted light 21a from the emitting portion 38 is shifted in the direction perpendicular to the optical axis 30 of the fiber projection tube 28, the boundary between the sample (low refractive index side) 2 and the cover glass 3 It is possible to generate evanescent light 4 that exudes within a range of several hundred nm from the surface.
[0021]
The fluorescent substance in the sample 2 existing in the vicinity of the surface of the cover glass 3 where the evanescent light 4 is generated is excited by the evanescent light 4 as excitation light to emit fluorescence, passes through the objective lens 7 and the dichroic mirror 11a, After the detrimental light in the wavelength region other than the fluorescence is removed by the absorption filter 12a, the light is guided to the observation imaging system 15 and imaged on the high-sensitivity camera (CCD or the like) 16, and the fluorescent substance in the sample 2 Can be observed.
[0022]
On the other hand, in the case of normal epi-illumination, as shown in FIG. 2, the high-reflection mirror 24 is removed from the optical axis 19 of the epi-illumination projection tube 17, and the fluorescence mirror unit 10a is replaced with the epi-fluorescence illumination fluorescent mirror unit 10b. Switch. In this case, the fluorescent mirror unit 10b includes a dichroic mirror 11b, an absorption filter 12b, and an excitation filter 13b. Of the light from the mercury burner 26 of the mercury lamp house 25, only the excitation light is transmitted through the excitation filter 13b, and the dichroic. Light reflected from the mirror 11 b and emitted from the tip of the objective lens 7 enters the sample 2 from the cover glass 3. Then, the fluorescence from the fluorescent material in the sample 2 passes through the dichroic mirror 11b, and after the detrimental light in the wavelength region other than the fluorescence is removed by the absorption filter 12b, it is guided to the observation imaging system 15. The image is formed on the high-sensitivity camera (CCD or the like) 16 and the fluorescent substance in the sample 2 can be observed.
[0023]
Next, specific embodiments will be described.
[0024]
(First embodiment)
3A, 3B, and 3C show a schematic configuration of a total reflection fluorescence microscope to which the first embodiment of the present invention is applied. In FIG. 3, the same parts as those in FIG.
[0025]
In this case, the epi-illumination projection tube 17 is fixed to an inverted microscope main body (not shown). The incident light projection tube 17 has a connection portion 17 a to the fiber projection tube 28 and a connection portion 17 b to the mercury lamp house 25 that holds the mercury burner 26. The optical axis 30 of the fiber projection tube 28 is perpendicular to the optical axis 19 of the epi-illumination projection tube 17, and the optical axis 27 of the mercury lamp house 25 is coincident with the optical axis 19 of the epi-illumination projection tube. Each is connected.
[0026]
Inside the epi-illumination projection tube 17, a high-reflection mirror 24 is fixed to the mirror holding part 22 by bonding or the like so as to reflect the parallel light 21 b of the fiber projection tube 28 onto the optical axis 19 of the epi-illumination projection tube 17. ing. The mirror holding part 22 is provided with an ant part 22a. The dovetail portion 22a is held in the dovetail groove portion 20 provided in the incident light projection tube 17 so as to be movable in the direction perpendicular to the paper surface, and the operation knob 23 is taken in and out of the incident light projection tube 17 from the outside in the paper surface vertical direction. Thus, the high reflection mirror 24 can be moved in the direction perpendicular to the paper surface. Further, the mirror holding part 22 is provided with a light shielding part 22b on the side surface on the mercury lamp house 25 side.
[0027]
The fiber projecting tube 28 includes a collimating lens 29 and a fiber introducing portion 32. A fiber introducing portion 32 is connected to an end portion of the fiber projecting tube 28 opposite to the incident light projecting tube 17 side.
[0028]
On the other hand, the emitted light from the laser light source 41 is incident on the optical fiber 39 from the optical fiber incident portion 40, and the emitted light 21 a is emitted from the optical fiber emitting portion 38. The optical fiber emitting portion 38 is fixed to the moving portion 37 with screws or the like (not shown). The outer side surface portion 37a of the moving portion 37 is fitted with the inner side surface portion 32a1 of the fiber introducing portion 32 and is movable in the left-right direction 54 on the paper surface.
[0029]
Here, the fiber introduction part 32 has a screw hole 32a having a center line 36 and a fitting hole 32b in parallel with the horizontal direction 54 of the moving part 37 in the drawing, and the screw hole 32a has a screw part. The lid cylinder 33 is engaged, and the adapter 35 is fitted in the fitting hole 32b.
[0030]
A compression coil spring 34, which is an elastic body, is sandwiched between the lid cylinder 33 and the adapter 35 in a state shorter than the natural length, and the adapter 35 abuts on the outer side surface portion 37a of the moving portion 37. It is like that. On the other hand, a slope contact portion 37 b is provided on the opposite side of the outer surface portion 37 a of the moving portion 37.
[0031]
A micrometer holding portion 42 is fixed to the fiber introducing portion 32 with screws or the like (not shown). A micrometer body 44 is held by the micrometer holder 42 with screws or the like (not shown).
[0032]
In the micrometer main body 44, a knob 46 is engaged with a rotating portion 44a with a screw or the like (not shown). The fiber introduction portion 32 is provided with a screw hole 32a and a cylindrical hole 32c having a center line orthogonal to the center line of the fitting hole 32b. The capsule adapter 43 is arranged in a state sandwiched between the contact portion 37 b and the tip end portion 44 b of the rotating portion 44 a of the micrometer body 44.
[0033]
As a result, the knob 46 of the micrometer main body 44 is rotated, the slope abutting portion 37b of the moving portion 37 is pressed by the capsule adapter 43 of the distal end portion 44b of the rotating portion 44a, and the moving portion 37 is pressed by the compression coil spring 34. By moving against the pressure, the incident angle of incident light emitted from the tip of the objective lens 7 and incident on the sample (low refractive index side) 2 from the cover glass (high refractive index side) 3 can be adjusted. The optical axis 31 of the outgoing light 21a from the optical fiber emitting portion 38 can be shifted from the optical axis 30 of the fiber projection tube 28 (to the left in the figure).
[0034]
The fixing portion 44c of the micrometer main body 44 is fitted with an opening 45a of a notch stopper 45 having an opening 45a and a U-shaped notch 45c as regulating means as shown in FIG. Has been. And the screw | thread 47 which adjusts the space | interval of the U-shaped notch part 45c is provided.
[0035]
Next, the operation of the first embodiment configured as described above will be described.
[0036]
In this case, the adapter 35 is in a state in which the outer side surface portion 37a of the moving portion 37 is pushed by the compression coil spring 34. It can be moved in the vertical direction 55, and the moving unit 37 can be moved in the horizontal direction 54 on the paper surface via the capsule adapter 43. Thereby, the optical axis 31 of the outgoing light 21a from the optical fiber emitting portion 38 is in a state parallel to the optical axis 30 of the fiber projector tube 28 and perpendicular to the optical axis 30 of the fiber projector tube 28. Can be adjusted.
[0037]
At the same time, the incident light angle emitted from the tip of the objective lens 7 to the sample 2 from the cover glass 3 can be adjusted. Here, the incident angle from the cover glass 3 to the sample 2 is critical. The position of the moving part 37 is adjusted by rotating the knob 46 so that it is slightly larger than the corner, and the side part 45d of the notch stopper 45 is brought into contact with the rotating part 44a of the micrometer body 44 at that position. 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, so that the notch stopper 45 is fixed to the micrometer main body 44. Position and fix against.
[0038]
Therefore, after that, the moving part 37 cannot move the optical fiber emitting part 38 to the optical axis 30 side of the fiber projection tube 28 due to the restriction of the notch stopper 45, and the cover glass 3 moves to the sample 2. Is restricted to movement only within the range of total reflection illumination, where the incident angle of the light is greater than the critical angle.
[0039]
On the other hand, the light beam (not shown) from the mercury lamp house 25 is shielded by the light shielding part 22b of the mirror holding part 22. By removing from the light beam, a light beam (not shown) from the mercury burner 26 can be guided to the epi-illumination projection tube 17. In this case, by rotating the mirror unit turret 9 around the rotation axis 14, the fluorescent mirror unit 10b including the excitation filter 13b, the dichroic mirror 11b, and the absorption filter 12b is arranged on the optical axis, thereby allowing a normal Epi-illumination can be observed.
[0040]
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, the incident angle is set to the critical angle. Since it is restricted within a larger range, fluorescence observation can be performed only with total reflection illumination. For this reason, it can prevent that the whole sample fades with the strong light by epifluorescence illumination, and can obtain the stable fluorescence observation by total reflection illumination. Moreover, since the incident angle from the cover glass 3 to the sample 2 can always be adjusted within the range of total 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, when switching from epi-illumination illumination to total reflection illumination with the illumination light applied to the sample (or vice versa). ), The cause of fading of the sample can also be avoided.
[0041]
In the first embodiment, the inverted microscope has been described, but the same effect can be obtained when applied to an upright microscope.
[0042]
(Modification of the first embodiment)
Next, a modification of the first embodiment will be described. 4 (a) and 4 (b) illustrate a modification of the first embodiment. Components identical with those shown in FIG.
[0043]
In this case, the fiber projection tube 28 is provided with a slide opening 28a on the rear side (the incident light projection tube 17 side) of the collimating lens 29, and the slide opening 28a has a slider having an opening 48a and an opening 48b. 48 is provided to be movable in the left-right direction 54 of the drawing. In the slider 48, a diffusion plate 49 is fixed to an opening 48a by a ring screw (not shown).
[0044]
On the other hand, the mirror holding portion 22 of the high reflection mirror 24 is directly fixed to the fixing portion 50 of the incident light projection tube 17 with screws or the like (not shown).
[0045]
With such a configuration, when the opening 48b of the slider 48 is arranged on the optical axis 30 of the fiber projection tube 28 in a state where the total reflection illumination is set as described in the first embodiment described above. Although this state is maintained, when the slider 48 is moved and the opening 48a having the diffuser plate 49 is arranged on the optical axis 30, the parallel light 21b is diffused and switched to epi-illumination.
[0046]
In this way, in addition to selecting the same effect as in the first embodiment described above, switching between total reflection illumination and epi-fluorescence illumination can be realized without using the mercury lamp house 25. .
[0047]
In the modification of the first embodiment, if the fiber light projection tube 28 is provided coaxially with the epi-illumination light projection tube 17, the high reflection mirror 24 can be omitted, and a more inexpensive structure can be achieved. can do.
[0048]
(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.
[0049]
In this case, the ant holding part 51 is provided in the mirror holding part 22 of the high reflection mirror 24, and this ant part 51 can be moved in the lateral direction 54 on the paper surface in the ant groove part 52 provided in the incident light projection tube 17. So that it is held.
[0050]
In this case, the mirror holding portion 22 is always in a state where the contact portion 51 a of the dovetail portion 51 is pushed by the compression coil spring 34 via the adapter 35. On the other hand, a micrometer holding portion 53 is provided on the side surface of the incident light projection tube 17 opposite to the position where the adapter 35 is disposed, and the micrometer main body 44 is provided in the micrometer holding portion 53. In this case, in the micrometer main body 44, the tip end portion 44 b of the rotating portion 44 a rotated by the rotation of the knob 46 is brought into contact with the contact portion 51 b of the dovetail portion 51.
[0051]
In this state, by rotating the knob 46 of the micrometer body 44, the ant portion 51 moves the mirror holding portion 22 along the reflection light path of the high reflection mirror 24, and the parallel light 21b is reflected by the high reflection mirror 24. The position can be adjusted.
[0052]
Thereby, the reflected light 21c of the parallel light 21b at the high reflection mirror 24 can be shifted from the optical axis 19, and the incident light angle emitted from the objective lens 7 to the sample 2 from the cover glass 3 can be adjusted. it can.
[0053]
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, a notch stopper is provided. 45 is applied to the rotating portion 44a of the micrometer main body 44, and the notch stopper 45 is fastened to fix the positioning to the micrometer main body 44.
[0054]
As a result, thereafter, the mirror holding unit 22 cannot move the reflected light 21c from the high reflection mirror 24 to the optical axis 19 side due to the restriction of the notch stopper 45, and the cover glass 3 to the sample 2 cannot be moved. It is restricted to movement only in the range of total reflection illumination where the incident angle is larger than the critical angle.
[0055]
In this case, similarly to the modification of the first embodiment, the laser light source 41 is provided by diffusing the parallel light 21b by providing the slider 48 and disposing the opening 48a having the diffusion plate 49 on the optical axis 30. It is also possible to obtain epi-fluorescent illumination using
[0056]
In the second embodiment, the mirror holding unit 22 is moved along the reflection optical path of the high reflection mirror 24. However, the same effect can be obtained by moving the mirror holding unit 22 along the incident optical path of the high reflection mirror 24. Can be obtained.
[0057]
In the above-described embodiment, the micrometer body 44 and the notch stopper 45 that restricts the movement of the rotating portion 44a of the micrometer body 44 are used. Instead of using these, the fiber light projecting tube 28 is used. A light-shielding plate having a slit hole shifted to the left or right from the center of the optical axis 30 is disposed in front of the optical fiber emitting portion 38, and the optical axis of the outgoing light emitted from the optical fiber emitting portion 38 is projected to the fiber. Even if it is moved to the optical axis 30 side of the tube 28, if the light on the optical axis center side of the emitted light is blocked by the light shielding plate, switching to the epi-illumination is prevented, and only in the range of the total reflection illumination. Can be restricted to the movement of. In this case, switching between the total reflection illumination and the epi-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 described in the first embodiment. Can do.
[0058]
Next, a method for realizing total reflection illumination will be described. The confirmation method when the incident angle of the illumination light from the cover glass 3 to the sample 2 exceeds the critical angle is to use an eyepiece (not shown) attached to a lens barrel (not shown) as a CT (centering telescope). In place of (not shown), a lens group (not shown) in the vicinity of the rear focal position 6 of the objective lens 7 is viewed using this CT. Illumination light is transmitted through a lens group (not shown) of the objective lens 7, and autofluorescence of the lens group occurs, and a bright spot can be observed in the lens group near the rear focal position 6 where light is particularly condensed.
[0059]
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 larger than the critical angle. In this case, two bright spots are observed symmetrically around the optical axis near the inside of the outer periphery of the rear focal position 6 of the objective lens 7. 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 from the tip of the objective lens 7 into the objective lens 7 again.
[0060]
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 larger state, the first bright spot gradually moves from the center side of the optical axis of the objective lens toward the outer peripheral direction, When the incident angle exceeds the critical angle, a second bright spot appears symmetrically with the optical axis center of the objective lens, and the notch stopper 45 is fixed to the micrometer main body 44 when the second bright spot appears.
[0061]
Next, the definition of near-total reflection illumination and the action and effect when applied to the first embodiment will be described based on the first example. Since the configuration is the same as that of the first embodiment, a description thereof will be omitted. Also, with respect to the function and effect, the same parts as those in the first embodiment are omitted, and only different parts will be described.
[0062]
First, near-total reflection illumination is defined as follows. In the total reflection illumination, the evanescent light 4 is generated in the range of about several hundreds of nanometers on the sample 2 side which is the low refractive index medium side at the boundary surface between the cover glass 3 and the sample 2. When the critical angle is made slightly smaller, refracted light from the cover glass 3 to the sample 2 is emitted from the cover glass 3 along the vicinity of the boundary surface of the sample 2. In this illumination method, a range of several μm in the vicinity of the cover glass 3 in the sample 2 can be illuminated. This is a kind of dark field illumination, and in the present invention, this illumination method is called near total reflection illumination.
[0063]
Next, the operation of near total reflection illumination will be described. Here, only near-total reflection illumination by the laser light source 41 is described, and description of epi-illumination illumination using the mercury burner 26 as a light source is omitted. First, as in the first embodiment, the knob 46 of the micrometer 44 is rotated to obtain 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 main body 44 is gradually rotated in the direction in which the incident angle from the cover glass 3 to the sample 2 is reduced, and adjusted to the illumination light that becomes the above-mentioned near-total reflection illumination.
[0064]
In this state, similarly to the first embodiment, the notch stopper 45 is positioned and fixed with respect to the micrometer main body 44, so that the optical fiber emitting portion 38 is limited only to the range of near total reflection illumination and total reflection illumination. Restrict movement.
[0065]
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 in addition to preventing fading of the entire sample 2 as in the first embodiment, the illumination light is emitted. It is possible to quickly change to total reflection illumination by moving away from the axis.
[0066]
In addition to the effects described above, it is possible to observe a region that cannot be observed with the evanescent light 4 generated by the total reflection illumination and the sample 2 that cannot be observed because the generated fluorescence is too weak. Further, in the epi-illumination illumination using the mercury burner 26 as the light source, the fluorescence is emitted from the entire sample 2, whereas in the near-total reflection illumination, the illumination range can be limited to a range of several μm near the cover glass 3, which is unnecessary. Fluorescence can be removed and observation with less background noise becomes possible.
[0067]
In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.
[0068]
Furthermore, the above 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 requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0069]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a total reflection fluorescent microscope capable of realizing stable fluorescence observation by total reflection illumination and near total reflection illumination and improving operability.
[Brief description of the drawings]
FIG. 1 is a schematic 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 fluorescent epi-illumination of a total reflection fluorescent microscope according to the present invention.
FIG. 3 is a diagram showing a schematic configuration of the 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 ... Rotating shaft
15 ... Observation imaging system
16 ... High sensitivity camera
17 ... Epi-illumination tube
17a ... Connection part
17b ... connection part
18 ... Condensing lens
19: Optical axis
20 ... Ant groove
21a: outgoing light
21b ... Parallel light
21c ... Reflected light
22 ... Mirror holding part
22a ... Ant part
22b ... Light-shielding part
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 part
32a1 ... Inner side surface
32a ... Screw hole
32b ... fitting 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 portion
39: Optical fiber
40: Optical fiber entrance
41 ... Laser light source
42 ... Micrometer holding part
43 ... Capsule adapter
44 ... Micrometer body
44a ... rotating part
44b ... tip
44c ... fixed part
44aa ... contact part
45 ... Notch stopper
45a ... opening
45c ... Notch
45d ... side surface
46 ... Knob
47 ... Screw
48 ... Slider
48a, 48b ... opening
49 ... Diffuser
50. Fixing part
51 ... Ant part
51a, 51b ... contact part
52 ... Ant groove
53 ... Micrometer holding part
54 ... right and left direction on paper
55 ... Up and down direction

Claims (4)

Via a light source and an illumination optical system and the objective lens, and a moving portion that is included in the illumination optical system Ru changing the incident angle of the illumination light irradiated onto the sample, total internal reflection illumination and near by moving the moving portion In the total reflection fluorescence microscope that enables switching of total reflection illumination,
A fine movement mechanism for finely moving the moving section, an operation section for operating the fine movement mechanism, and a regulating means for regulating a movement range of the moving section,
The range of movement, total internal reflection fluorescence microscopy, wherein the incident angle to the sample through the objective lens of the illumination light is in a range of total internal reflection illumination and near total internal reflection illumination is obtained. The illumination optical system has an optical fiber, the moving part, the exit end of the optical fiber, movably disposed et al is in a direction perpendicular to the optical axis,
The restricting means restricts a moving range in a direction perpendicular to an optical axis of an output end of the optical fiber by operation of the operation unit to a range in which the total reflection illumination and near total reflection illumination can be obtained. The total reflection fluorescent microscope according to claim 1. The illumination optical system includes a reflecting member, and the moving unit moves the moving member along an incident optical path of illumination light to the reflecting member, or an optical axis direction of the reflected light path,
The regulating means, total reflection of claim 1, wherein the restricting a range in which the incident light path, or the total reflection illuminated and near total internal reflection illumination of the movement range along the optical axis direction of the reflected light path is obtained Fluorescence microscope. Total internal reflection fluorescence microscope according to claim 1 or 3 further characterized in that the optical elements for diffusing the illumination light to the optical path of the illumination optical system provided so as to be inserted and removed.

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