JP4331425B2 - Microscopic total reflection illuminator - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a total reflection illumination device for a microscope that illuminates a specimen through an objective lens having a numerical aperture capable of total reflection illumination.
[0002]
[Prior art]
In recent years, as a biological microscope used for fluorescence observation, a microscope called a total reflection fluorescence microscope (TIRFM: Total Internal Reflection Fluoro-response Microscopy) has attracted attention.
[0003]
In this microscope, when the illumination light is totally reflected at the boundary surface between the cover glass and the specimen, the fluorescent material is excited using light called evanescence light that oozes in a slight range of several hundred mm or less on the specimen side. Since only a small range of fluorescence in the vicinity of the cover glass is observed, the background is very dark and weak fluorescence can be observed.
[0004]
Therefore, conventionally, in such a microscope, as a device that performs total reflection illumination through an objective lens, as shown in FIG. 6 (Japanese Patent Laid-Open No. 09-159922), the illumination light 102 is reflected in the direction of the objective lens 100. The mirror 101 for moving is provided so as to be movable, and by moving the mirror 101, the incident position of the illumination light 102 on the objective lens 100 is moved in a direction away from the optical axis of the objective lens 100. As shown in FIG. 7 (Japanese Patent Laid-Open No. 10-96861), the frame of the objective lens 200 has a double structure so that the angle of the light beam emitted to the glass 103 side is changed. The light is reflected by the annular mirror 202, and the sample 203 is irradiated with illumination light between the inner frame and the outer frame of the double structure frame. That so as to observe the light through the objective lens 204 is considered.
[0005]
[Problems to be solved by the invention]
By the way, in the total reflection illumination through the objective lens, the illumination light applied to the specimen returns in principle to the objective lens again. Further, a mirror for guiding the illumination light to the objective lens side is provided in the observation light path from the objective lens to the observation means.
[0006]
Therefore, in both of the cases shown in FIGS. 6 and 7, the observation light path of the fluorescence emitted from the specimen intersects with the illumination light and the totally reflected return light. The autofluorescence generated on the returned light beam enters the observation light beam, which may deteriorate the fluorescence observation image.
[0008]
The present invention has been made in view of the above circumstances, and an object thereof is to provide a total reflection illumination device for a microscope that can always ensure a good fluorescence observation image.
[0009]
[Means for Solving the Problems]
The invention according to claim 1 is a total reflection illumination device for a microscope that illuminates a specimen with illumination light from a light source via an objective lens having a numerical aperture capable of total reflection illumination. A first total reflection mirror that is disposed in the vicinity of the outermost peripheral portion of the observation optical path passing through the ball lens, and that reflects the incident illumination light in a direction along the observation optical path of the objective lens and toward the front lens. The specimen and the objective of the specimen are disposed at a position symmetrical to the first total reflection mirror with respect to the observation optical axis of the objective lens and in the vicinity of the outermost peripheral portion of the observation optical path of the objective lens. A return that is totally reflected at the boundary surface with the cover glass disposed on the lens side, passes along the observation optical path of the objective lens, and passes through the front lens in the internal space of the objective lens. In front of light A second total reflection mirror that reflects in a direction different from the observation optical path and away from the observation optical path, and a return light dimming means that attenuates the return light reflected by the second total reflection mirror; It is characterized by comprising.
[0010]
According to a second aspect of the present invention, in the first aspect of the present invention, the return light dimming means has an external installation section installed outside the microscope.
[0011]
According to a third aspect of the present invention, in the second aspect of the present invention, the return light attenuating means is connected to the external installation portion via an optical fiber.
[0012]
The invention according to claim 2 is the invention according to any one of claims 1 to 3, wherein the first total reflection mirror and the second total reflection mirror can be inserted into and removed from the observation optical path of the objective lens . It is characterized by being installed on a support member.
[0013]
According to a fifth aspect of the present invention, in the fourth aspect of the present invention, the microscope or the support member has switching means for turning on / off illumination light in conjunction with insertion / removal of the support member. It is said.
[0014]
As a result, according to the present invention, the fluorescence observation light beam emitted from the specimen does not intersect the illumination light beam reflected by the first total reflection mirror and the return light beam incident on the second total reflection mirror on the observation light path. It is also possible to prevent the illumination light beam irradiated on the specimen and the return light beam after total reflection from intersecting on the observation optical path.
[0015]
In addition, according to the present invention, the return light beam reflected by the second total reflection mirror is positively excluded from the outside of the microscope, so that the possibility of the scattered light of the return light beam entering the observation optical path can be further reduced. .
[0016]
Furthermore, according to the present invention, since the illumination light can be turned on / off in conjunction with the insertion / removal of the support member, the illumination light can be automatically stopped when the support member is removed.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0018]
(First embodiment)
FIG. 1 shows a schematic configuration of a total reflection illumination device of a microscope to which the first embodiment of the present invention is applied.
[0019]
In the figure, reference numeral 1 denotes an objective lens revolver holding base, and this objective lens revolver holding base 1 is attached to a microscope body (not shown).
[0020]
The objective lens revolver holder 1 holds a fixed portion 2a of the objective lens revolver 2. A rotating portion 2b is rotatably provided on the fixed portion 2a of the objective lens revolver 2. A plurality of objective lenses 3 (only one is shown in the illustrated example) are fixed to the rotating unit 2b with screws. By rotating the rotating unit 2b, the desired objective lens 3 is placed on the observation optical axis. It can move on O. In this case, circular holes 1a and 2a1 around the observation optical axis O passing through the center of the objective lens 3 are provided in the fixed part 2a of the objective lens revolver holding base 1 and the objective lens revolver 2, respectively. .
[0021]
The objective lens 3 has a numerical aperture capable of total reflection illumination, and has a front lens 3a at the tip, and the immersion oil 5 is located at a position where the front lens 3a faces the cover glass 4 on which the sample S is placed. Is arranged through.
[0022]
A concave portion 1 b is formed on the upper surface of the objective lens revolver holder 1. The concave portion 1b is provided with a box-shaped slider main body 6 as a support member so as to be inserted / removed along a plane orthogonal to the observation optical axis O. When the slider body 6 is mounted in the concave portion 1b of the objective lens revolver holding base 1, holes 6a and 6b through which the observation optical path a passes are formed on the upper and lower surfaces positioned on the observation optical axis O, respectively. Yes.
[0023]
An emission end of the optical fiber 7 is fixed to the side surface of the slider body 6 via an optical fiber connector 8.
[0024]
A convex lens 9 is disposed in the slider body 6 at a position facing the emission end of the optical fiber 7, and the first total reflection mirror 10 is disposed on the optical path of the light that is transmitted through the convex lens 9 and converged. Has been placed.
[0025]
In this case, the convex lens 9 converts the diffused light emitted from the optical fiber 7 into a convergent light, and the light reflected in the direction of the objective lens 3 by the first total reflection mirror 10 is the rear focal point of the objective lens 3. It has a focal length that focuses at the position 3b.
[0026]
The first total reflection mirror 10 is disposed in the vicinity of the outermost peripheral portion of the observation optical path a, reflects the convergent light beam transmitted through the convex lens 9 in the direction of the objective lens 3, and reflects the reflected light inside the objective lens 3. The vicinity of the outermost peripheral portion of the space is advanced along the observation optical axis O.
[0027]
On the other hand, the second total reflection mirror 11 is arranged in a symmetric positional relationship with respect to the first total reflection mirror 10 and the observation optical axis O. The second total reflection mirror 11 reflects the return light reflected by the sample S surface and returned near the outermost peripheral portion of the internal space of the objective lens 3 along the observation optical axis O in a direction different from the observation optical path a. I have to.
[0028]
In the reflection direction of the second total reflection mirror 11, an optical trap 12 is arranged as a return light dimming means. The optical trap 12 reduces the return light reflected by the second total reflection mirror 11.
[0029]
As shown in FIG. 2, the first total reflection mirror 10 is provided with a first guide shaft 13. The first guide shaft 13 has a straight portion 13a and a bent portion 13b obtained by bending the tip portion of the straight portion 13a at a substantially right angle, and the first total reflection mirror 10 is attached to the bent portion 13b. Yes. Further, the first guide shaft 13 is supported by the pair of guides 14a and 14b with the straight portion 13a, and moves linearly along the guides 14a and 14b so that the first total reflection mirror 10 is moved along the observation optical axis O. Can be moved closer to and away from.
[0030]
The second total reflection mirror 11 is provided with a second guide shaft 15 disposed in parallel with the first guide shaft 13. The second guide shaft 15 has a straight portion 15a and a bent portion 15b obtained by bending the tip portion of the straight portion 15a at a substantially right angle, and the second total reflection mirror 11 is attached to the bent portion 15b. Yes. The second guide shaft 15 is supported by the pair of guides 16a and 16b on the straight portion 15a, and moves linearly along the guides 16a and 16b, thereby moving the second total reflection mirror 11 to the observation optical axis O. Can be moved closer to and away from.
[0031]
A link 17 is provided between the first guide shaft 13 and the second guide shaft 15. The link 17 is rotatably supported at the intermediate portion on the bottom surface of the slider body 6, and both ends are rotatable at the straight portion 13 a of the first guide shaft 13 and the straight portion 15 a of the second guide shaft 15. The second guide shaft 15 is attached and moved in the direction opposite to the moving direction of the first guide shaft 13 with respect to the movement of the first guide shaft 13.
[0032]
The link 17 is provided with a spring 18 between the bottom surface of the slider body 6. The spring 18 always applies a counterclockwise rotational torque to the link 17.
[0033]
An operation shaft 19 is provided on a side surface of the slider body 6 through a screw portion 19a. The operation shaft 19 is in contact with the end portion of the first guide shaft 13, and by adjusting the screwing amount of the screw portion 19a by operating the knob 19b, the first guide shaft 13 is moved linearly. The first total reflection mirror 10 and the second total reflection mirror 11 can be moved closer to and away from the observation optical axis O while maintaining a symmetrical positional relationship with respect to the observation optical axis O.
[0034]
Next, the operation of the first embodiment configured as described above will be described.
[0035]
Now, when light is emitted from the emission end of the optical fiber 7, it becomes diffused light and enters the convex lens 9. Then, it is converted into a convergent light beam by the convex lens 9, reflected in the direction of the objective lens 3 by the first total reflection mirror 10, and travels along the observation optical axis O in the vicinity of the outermost peripheral portion of the internal space of the objective lens 3. Then, the light is focused at the back focal position 3b of the objective lens 3, and again becomes a diffused light beam, passes through the front lens 3a, and then is emitted from the objective lens 3 as a parallel light beam inclined with respect to the observation optical axis O. Then, the specimen S is irradiated through the immersion oil 5 and the cover glass 4.
[0036]
Here, when the operating shaft 19 is rotated by the operation of the knob 19b, the first guide shaft 13 and the second guide shaft 15 are linearly moved in the opposite directions in accordance with the screwing amount of the screw portion 19a, and the first whole shaft is moved. The reflection mirror 10 and the second total reflection mirror 11 are adjusted in the contact / separation direction with respect to the observation optical axis O while maintaining a symmetrical positional relationship with respect to the observation optical axis O. The distance of the light beam traveling in space from the observation optical axis O changes, and the tilt angle of the parallel light beam emitted from the objective lens 3 changes. That is, by rotating the knob 19b of the operation shaft 19 to change the inclination angle of the parallel light beam emitted from the objective lens 3, the total reflection illumination can be obtained by setting the inclination angle to be equal to or greater than the critical angle.
[0037]
On the other hand, the light beam totally reflected by the sample S surface is incident on the objective lens 3 again as return light, and after being focused at the back focal position 3b of the objective lens 3, is totally reflected by the second total reflection mirror 11. Introduced into the optical trap 12 and terminated.
[0038]
In this state, when fluorescence is emitted from the sample S, this fluorescence travels along the observation optical path a as the observation light beam O ′ along the observation optical axis O. At this time, the observation light beam O ′ is the first light beam. The illumination light beam reflected by the total reflection mirror 10 and the return light beam incident on the second total reflection mirror 11 are guided to the observation side without intersecting. Further, the illumination light beam irradiated on the specimen S and the return light beam after total reflection do not intersect on the observation light path a and are excluded from the observation light path a.
[0039]
Therefore, according to such a configuration, the fluorescent observation light beam O ′ emitted from the specimen S is incident on the illumination light beam reflected by the first total reflection mirror 10 or the second total reflection mirror 11 on the observation light path a. Therefore, it is possible to prevent the autofluorescence generated on the illumination light beam and the return light beam from entering the observation light beam O ′, and to greatly suppress the deterioration of the fluorescence observation image. . Further, since the illumination light beam applied to the specimen S and the return light beam after total reflection do not intersect on the observation light path a, for example, even when laser light is used as illumination light, the laser beams intersect each other. Interference fringes caused by this can be prevented, and a good fluorescence observation image can always be secured.
[0040]
In the first embodiment described above, the first total reflection mirror 10 and the second total reflection mirror 11 are constituted by the first guide shaft 13, the second guide shaft 15 and the link 17 as moving means. However, the present invention can also be implemented using other moving means such as an electric motor. Further, although the convex lens 9 is fixed, it can be moved along the illumination light beam so that precise optical adjustment is possible.
[0041]
(Second Embodiment)
Next, a second embodiment of the present invention will be described.
[0042]
FIG. 3 shows a schematic configuration of the second embodiment of the present invention, and the same parts as those in FIG.
[0043]
In this case, the convex lens 21 is disposed on the reflection optical path of the second total reflection mirror 11. This convex lens 21 is for converging the return light totally reflected by the second total reflection mirror 11, and the incident end 22a of the optical fiber 22 is located at the condensing position of the return light by the convex lens 21. It is positioned. In this case, the incident end 22a of the optical fiber 22 has a sufficiently large core diameter so that light can be reliably introduced into the fiber even if the return light condensing position is slightly shifted. An optical trap 23 is connected to the emission end 22 b of the optical fiber 22. This optical trap 23 is installed outside the microscope.
[0044]
Next, the operation of the second embodiment configured as described above will be described.
[0045]
In this case, the light beam totally reflected by the sample S surface is incident again on the objective lens 3 as return light, and after being focused at the back focal position 3b of the objective lens 3, is totally reflected by the second total reflection mirror 11. And enters the convex lens 21. Then, the light passes through the convex lens 21, becomes a convergent light beam again, enters the incident end 22 a of the optical fiber 22, and terminates at the optical trap 23 outside the microscope.
[0046]
Accordingly, in this way, the same effect as described in the first embodiment described above can be obtained, and the return light reflected by the second total reflection mirror 11 using the optical fiber 22 can be positively reflected. Therefore, the possibility that the scattered light of the return light beam enters the observation optical path a can be extremely reduced, and a more favorable fluorescence observation image can be secured.
[0047]
(Third embodiment)
Next, a third embodiment of the present invention will be described.
[0048]
4 and 5 show a schematic configuration of the third embodiment of the present invention. The same parts as those in FIG. 2 are denoted by the same reference numerals.
[0049]
In this case, the slider main body 6 detachably provided in the recess 1b of the objective lens revolver holding base 1 is provided with a pair of click grooves 31 and 32 that are separated from each other by a predetermined distance along the side surface in contact with the recess 1b. Yes. A ball plunger 33 is provided on the side of the objective lens revolver holder 1 on the side of the concave portion 1 b where the slider body 6 is in contact.
[0050]
The ball plunger 33 always presses the side surface of the slider body 6 and positions the slider body 6 by falling into the click grooves 31 and 32. The slider body 6 is positioned on the observation optical axis O as shown in FIG. When the slider body 6 is retracted from the observation optical axis O as shown in FIG. 5, it falls into the click groove 32.
[0051]
On the other hand, in the concave portion 1 b of the objective lens revolver holding base 1, a micro switch 34 is disposed at a position pressed by the tip of the slider body 6. As shown in FIG. 4, the microswitch 34 is pressed and conducted while the slider body 6 is inserted into the observation optical axis O.
[0052]
An interlock terminal 36 a of a laser oscillator 36 is connected to the output terminal of the microswitch 34 via a cable 35. The laser oscillator 36 oscillates when the interlock is released by the conduction of the microswitch 34 and generates a laser beam. The laser beam is introduced into the slider body 6 via the optical fiber 7. Yes.
[0053]
Next, the operation of the third embodiment configured as described above will be described.
[0054]
Now, as shown in FIG. 4, when the slider body 6 is inserted up to the observation optical axis O, the ball plunger 33 falls into the click groove 31, and the slider body 6 is held so as not to move easily. In this state, when the micro switch 34 is pressed and conducted by the tip of the slider body 6, the conduction of the micro switch 34 releases the interlock of the laser oscillator 36 and generates a laser beam. This laser beam is introduced into the slider body 6 through the optical fiber 7, and the same operation as described in the first embodiment is obtained.
[0055]
On the other hand, as shown in FIG. 5, when the slider body 6 is retracted from the observation optical axis O, the ball plunger 33 falls into the click groove 32, and the slider body 6 is held so as not to move easily. In this state, the microswitch 34 is opened, the interlock of the laser oscillator 36 connected to the cable 35 is activated, and no laser beam is generated.
[0056]
Therefore, even if it does in this way, the same effect as described in the first embodiment described above can be obtained, and furthermore, the case where the slider body 6 is inadvertently extracted from the concave portion 1b of the objective lens revolver holding base 1 In addition, since the laser beam from the laser oscillator 36 can be surely automatically stopped, an excellent safety effect can be obtained.
[0057]
In the third embodiment described above, the cable 35 is directly connected to the interlock terminal 36a of the laser oscillator 36. For example, an electric shutter is inserted between the laser oscillator 36 and the optical fiber 7, and the cable 35 is connected. The same effect as described above can be obtained by connecting to the electric shutter and using means such as interlocking the opening / closing operation of the electric shutter with the on / off operation of the micro switch 34.
[0058]
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.
[0059]
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.
[0060]
Each embodiment described above includes the following inventions.
[0061]
(1) A total reflection illumination device for a microscope that illuminates a specimen through an objective lens having a numerical aperture capable of total reflection illumination, and illumination light incident from the outside of the microscope is disposed in the vicinity of the outermost periphery of the observation optical path The return light reflected in the direction of the objective lens by the first total reflection mirror and the total reflection on the specimen surface is disposed in a positional relationship symmetrical to the first total reflection mirror with respect to the observation optical axis. A total reflection illumination device for a microscope, which is reflected by a second total reflection mirror and terminated by an optical trap means.
[0062]
(2) In the total reflection illumination device for a microscope according to (1), the light trap means is arranged outside the microscope through an optical fiber.
[0063]
(3) In the total reflection illuminating device for a microscope according to (1), the total reflection illuminating device for the microscope can be retracted integrally from the observation optical path, and the illumination light is linked to the insertion / retraction to the observation optical path. It is characterized by being turned on and off.
[0064]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a total reflection illumination device for a microscope that can always ensure a good fluorescence observation image.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.
FIG. 2 is a diagram showing a schematic configuration of a slider main body used in the first embodiment.
FIG. 3 is a diagram showing a schematic configuration of a second embodiment of the present invention.
FIG. 4 is a diagram showing a schematic configuration of a slider main body used in a third embodiment of the present invention.
FIG. 5 is a diagram showing a schematic configuration of a slider body used in a third embodiment.
FIG. 6 is a diagram showing a schematic configuration of an example of a device employing conventional total reflection illumination.
FIG. 7 is a diagram showing a schematic configuration of an example of a device employing conventional total reflection illumination.
[Explanation of symbols]
1 ... Objective lens revolver holder 1a. 2a1 ... hole 1b ... concave 2 ... objective lens revolver 2a ... fixed part 2b ... rotating part 3 ... objective lens 3a ... front lens 3b ... side focal position 4 ... cover glass 5 ... immersion oil 6 ... slider bodies 6a, 6b ... Hole 7 ... Optical fiber 8 ... Optical fiber connector 9 ... Convex lens 10 ... First total reflection mirror 11 ... Second total reflection mirror 12 ... Optical trap 13 ... First guide shaft 13a ... Linear portion 13b ... Bending portion 14a , 14b ... Guide 15 ... Second guide shaft 15a ... Linear portion 15b ... Bending portion 16a, 16b ... Guide 17 ... Link 18 ... Spring 19 ... Operation shaft 19a ... Screw portion 19b ... Knob 21 ... Convex lens 22 ... Optical fiber 22a ... Incident end 22b ... Exit end 23 ... Light trap 31, 32 ... Click groove 33 ... Ball plunger 34 ... Micro switch 35 ... Cable 36 Laser oscillator 36a ... interlock terminal

Claims (5)

In a total reflection illumination device of a microscope that illuminates a specimen with illumination light from a light source through an objective lens having a numerical aperture capable of total reflection illumination,
Arranged in the vicinity of the outermost periphery of the observation optical path that passes through the front lens in the internal space of the objective lens, and reflects the incident illumination light in the direction along the observation optical path of the objective lens and toward the front lens A first total reflection mirror that
The specimen and the objective lens of the specimen are arranged in a position symmetrical to the first total reflection mirror with respect to the observation optical axis of the objective lens and disposed in the vicinity of the outermost peripheral portion of the observation optical path of the objective lens. Return light that has been totally reflected at the boundary surface with the cover glass disposed on the side and returned in the direction along the observation optical path of the objective lens and through the front lens in the internal space of the objective lens A second total reflection mirror that reflects in a direction different from the observation optical path and away from the observation optical path ;
Return light dimming means for dimming the return light reflected by the second total reflection mirror;
A total reflection illumination device for a microscope, comprising:   2. The total reflection illumination device for a microscope according to claim 1, wherein the return light dimming means has an external installation part installed outside the microscope.   The total reflection illumination device for a microscope according to claim 2, wherein the return light attenuation means is connected to the external installation section via an optical fiber.   The first total reflection mirror and the second total reflection mirror are installed on a support member that can be inserted into and removed from the observation optical path of the objective lens. The total reflection illumination device of the microscope described.   5. The total reflection illumination device for a microscope according to claim 4, wherein one of the microscope and the support member has switching means for turning on / off illumination light in conjunction with insertion / removal of the support member.

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