JP2003131141A - Rotational zone total reflection illumination mechanism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rotational zone total reflection illumination mechanism in which an illumination without polarization even at the periphery of a visual field is given and a fluorescence information of all directions is available. SOLUTION: In a Koehler type illumination system in which an object is illuminated by introducing laser light to the peripheral part of an objective lens of a microscope, the rotational zone total reflection illumination mechanism is characterized in that the direction of illumination with the laser light is rotatable around the axis of the objective lens 56 of the microscope.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rotary annular total reflection illuminating mechanism in which an illuminating direction of a laser beam is rotatable in an illuminating system in which a laser beam is incident on a peripheral portion of a microscope objective lens, More specifically, the present invention relates to a rotary annular total reflection illumination mechanism suitable for an illumination system that emits fluorescence using total reflection light.

[0002]

2. Description of the Related Art The principle of total reflection using conventional evanescent illumination will be briefly described. FIG. 5 shows that a laser beam is made incident on the peripheral portion of a microscope objective lens to emit fluorescence using total reflection light. Total reflection lighting (evanescent lighting:
It is a principle view of a normal inner angle of 61 ° and an outer angle of 68 °.

In the drawing, 11 is an objective lens, 12 is oil immersion oil, 13 is glass, and 14 is a solution. When a laser beam is incident on the peripheral portion of the objective lens 11, it is totally reflected by the glass / aqueous solution portion and near the surface. Light exudes at a depth of 150 nm (this light field is called the evanescent field). When this evanescent field is used for fluorescent illumination, the background light is significantly reduced and a high-contrast fluorescent-molecular image can be obtained. However, there is a problem that only the fluorescence information that is biased can be obtained by total reflection illumination that illuminates from one direction using the evanescent field.

Further, FIG. 6 shows another example of the prior art, which is an illumination optical system for performing annular total reflection illumination with the central part shielded.
In FIG. 6, 20 is a sample surface, 21 is an objective lens, 22 is an objective lens pupil surface, 23 is a reflecting mirror, 24 is a convex lens, and 25
Is a field stop surface (object surface and conjugate surface), and 26 is a central light-shielding diaphragm (conjugate with the objective lens pupil surface 22). This illumination method adopts a method of converging a laser beam on the sample surface 20 (critical illumination). This is different from FIG. 1 in which the sample surface 20 is illuminated with a parallel beam. In the present illumination system, since the ring-shaped total reflection illumination is performed, the central light-shielding diaphragm 26 is provided on the conjugate plane with the objective lens pupil plane 22, and the conventional technique uses this illumination system.

However, this illumination method is used for the sample surface 20.
Since the laser beam is focused at the center, the brightness is different between the center and the periphery of the field of view, so that it is difficult to obtain uniform fluorescence information and it is difficult to expand the field of view. In addition, there is a drawback that the brightness is significantly reduced when a diffusion plate is inserted to expand the field of view.

By the way, general microscope illumination is ideally Koehler illumination which is bright and has no illumination unevenness. FIG. 7 shows the focus state of the object plane using Koehler illumination. In the figure, the laser beam must be focused at the focal position (front focal plane) F of the objective lens as shown in FIG. When a laser beam with excellent parallelism is used, 1o, 2 on the center optical axis of the object plane
Light rays from four directions of o, 3o, and 4o are condensed and become bright. However, the uppermost point S around the visual field is 1b and 3b.
Since only the rays of light are condensed and the periphery is not only dark but also polarized, it is difficult to perform uniform illumination including the polarization direction, and there is a problem that correct fluorescence information cannot be obtained.

In recent years, a minute probe (fluorescent dye-molecule)
It is possible to catch a dynamic movement here to know the specific direction of the protein using, and it will become an important issue in the future. However, in order to solve this problem, the total internal reflection illumination (evanescent illumination) that illuminates from one direction described above can obtain only biased fluorescence information, and the Koehler illumination type annular total internal reflection illumination can be said to be ideal illumination. However, due to the above-mentioned problems, there are no examples of adoption.

Further, the illumination angle of the total internal reflection illumination varies depending on the change in the refractive index of the medium surrounding the sample (corresponding to different medium, temperature change of the sample), and unless it is strictly handled, unnecessary background light is caused, and the single molecule fluorescent dye is produced. Is difficult to observe. Further, if the magnification (focal length) of the objective lens used changes, there is a troublesome problem of changing to different ones for the illumination system lens and the zonal aperture each time in order to cope with this.

[0009]

Therefore, according to the present invention,
By rotating (rotating) the laser beam along the edge of the objective lens, it is possible to illuminate unpolarized light even to the periphery of the field of view, and a rotary annular total reflection illumination mechanism that can obtain fluorescence information in all directions. It aims to solve the above problems. According to the rotatable total internal reflection illumination mechanism of the present invention, it is possible to know the direction in which the excitation efficiency of the sample or the fluorescent dye is high from the information on the maximum direction of the fluorescence brightness.
Also, by combining with a polarizing element such as an analyzer (analyzer), it is possible to know the specific direction of the protein using the fluorescent dye-molecules, and it is possible to individually capture the dynamic movement. Furthermore, by the tilt fine adjustment mechanism of the reflecting mirror,
In addition to being able to strictly correspond to the focal length of the objective lens used, it is possible to closely respond to changes in the refractive index of the medium surrounding the sample (correspondence to different media, temperature changes of the sample).

[0010]

Therefore, the solution adopted by the present invention is, in a Koehler illumination system for illuminating an object by introducing a laser beam to the peripheral portion of a microscope objective lens,
The rotary annular total reflection illumination mechanism is characterized in that the illumination direction by the laser beam can be rotated while being tilted about the axis of the microscope objective lens. Further, the illumination system is a rotary annular total reflection illumination mechanism characterized by being an illumination system which introduces a laser beam into a peripheral portion of a microscope objective lens and totally reflects it to emit fluorescence. Further, in order to rotate the illumination direction by the laser beam and give an optimum total reflection angle to the objective lens to be used, a reflecting mirror having a mechanism capable of finely adjusting the tilt of the reflecting mirror, and for rotating the reflecting mirror It is a rotary type annular total reflection illuminating device characterized by comprising a rotating means. Further, in the rotary annular total reflection illuminator, the rotation center of the rotating means is arranged at a position slightly apart from the rotation center axis of the reflecting mirror. Further, in order that the vibration due to the rotation of the reflecting mirror does not adversely affect the microscope, the rotating means including the reflecting mirror has a structure in which the shape and weight are symmetrical with respect to the center axis of rotation, and the whole rotary ring zone. It is a reflective lighting device.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS An optical system as an embodiment according to the present invention will be described below. FIG. 1 is a diagram of the entire optical system, FIG. 2 is a partial sectional view of a reflecting mirror, and FIG. 3 is A- in FIG. 4A and 4B are optical system diagrams in a state in which the tilt of the reflecting mirror is changed. In FIG. 1, 51 is a laser fiber light source, 52 is a collector lens, 53 is a front focal plane of the convex lens 56 (also coincides with the rear focal plane of the collector lens 52), 54 is a motor, 55 is a reflecting mirror, and 56 is a convex lens.
57 is a dichroic mirror, 58 is a front focal plane of the objective lens, 59 is an objective lens, and 60 is an object plane (sample plane).

Now, the structure of the reflecting mirror 55 will be described in detail with reference to FIGS. 2 and 3. In FIG.
Reference numeral 61 is a reflecting mirror body, 62 is a reflecting mirror body and other holding metal pieces, and 63 is a main body metal piece, all of which have a circular shape.
The reflecting mirror body 61 is fixed to the reflecting mirror holding metal member 62 by adhesion or the like. As shown in FIG. 3, pivot grooves 62a are provided at two symmetrical positions (on a straight line as a rotation axis) on the outer circumference of the reflecting mirror holding metal 62, and screw holes 63a formed at two outer circumferences of the main metal fitting 63. Pivot screw 6 attached to
6. The lock nut 67 is attached to the metal body 63 so as to be rotatable on the axis of the pivot screw 66.

Fine adjustment screws 64 are provided on the main metal part 63 at two symmetrical positions sandwiching the pivot screw shaft for fine adjustment of the tilt of the reflecting mirror 61. The structure is such that the tilt of the reflecting mirror body is finely adjusted by using the pivot screw 66 as an axis, and finally the fine adjustment screw 64 is tightened and fixed by the lock nut 65.

The body hardware is fixed to a rotary shaft 54a of a motor 54 as a rotating means at a position slightly apart from the center of the body hardware. This is because the reflecting mirror 55 in the optical system has a conjugate relationship with the object surface 60 (sample surface), and the rotation center of the motor 54 is set so that slight dust on the reflecting mirror surface does not form an image on the object surface 60 (sample surface). The shaft is installed with a slight shift. Further, as shown in FIGS. 2 and 3, the rotating body including the reflecting mirror is formed on the central axis of rotation so as to have a symmetrical shape and weight structure so that the vibration due to the rotation of the reflecting mirror does not adversely affect the microscope. There is.

In the optical system having the above-mentioned structure, the laser fiber light source 51 becomes a parallel light beam by the collector lens 52, but since it is a mechanism that rotates while the reflecting mirror 55 is tilted, it becomes a circular light beam and around the convex lens 56. The light incident on is condensed on the rear focal plane 58 of the convex lens 56 (the front focal plane of the objective lens 59).

When the reflecting mirror 55 is tilted by + α, the laser beam parallel to the optical axis is incident on the edge of the convex lens 56 with a double α tilt, as shown in FIG. The light becomes parallel to the axis and illuminates the focal plane of the objective lens 59 on the front side of the objective lens in a ring shape. When the reflecting mirror 55 is tilted by -α, the laser beam parallel to the optical axis is incident on the opposite edge of the convex lens 56 and parallel to the optical axis as shown in FIG. Then, the focal plane of the objective lens 59 on the front side of the objective lens 59 is illuminated like a ring.

The light thus condensed becomes a parallel light beam again by the objective lens 59 (the role of a condenser) and illuminates the object surface 60 (sample surface). As described above, the mechanism for finely adjusting the tilt of the reflecting mirror and the rotating mechanism for the reflecting mirror can not only strictly correspond to the focal length of the objective lens used, but also change the refractive index of the medium surrounding the sample (for different media). Correspondence, temperature change of sample) Also,
By rotating the reflecting mirror 360 ° by a motor, the sample surface can be illuminated from the surrounding 360 ° direction, and the problem of the total internal reflection illumination of the conventional evanescent illumination that illuminates from one direction, and further the problem of the conventional Koehler microscope It can surely solve the problems that it has. Further, since the laser beam has a very excellent coherence, an interference fringe of the illumination optical system is generated. In the case of fluorescence observation, the excitation light (illumination light) should be removed by a dichroic mirror or a filter, but the problem is that the excellent coherence becomes excitation light (illumination light) groups and a uniform fluorescence image cannot be obtained. There is. However, in the present optical system configured as described above, the rotating reflecting mirror is arranged on the sample surface and the optically conjugate plane, and even if this interference fringe occurs, the interference fringe is generated by the rotation of the reflecting mirror inclined to the optical axis. Is rotated and averaged, resulting in the same effect as removed.

In order to perform total reflection illumination, the tilt angle α of the reflecting mirror 55 may be adjusted so that light within 61 ° to 68 ° is sent to the object surface (sample surface). Further, it is desirable that the fiber serving as the laser fiber light source 51 has a small core diameter.

Although the embodiment of the present invention has been described above, the rotating mechanism of the reflecting mirror, the mechanism for finely adjusting the tilt of the reflecting mirror, and the like are not limited to those described above, and any similar function can be achieved. For example, other mechanisms can be adopted. The present invention can be embodied in any other form without departing from the spirit or the main features thereof. Therefore, the above-described embodiments are merely examples in all respects and should not be limitedly interpreted.

[0020]

According to the rotatable total internal reflection illumination mechanism of the present invention, even the peripheral portion of the visual field can be illuminated uniformly, and uniform fluorescence information in all directions can be obtained. In addition, the direction in which the excitation efficiency of the sample or the fluorescent dye is high can be known from the information on the maximum direction of the fluorescence brightness. Also, by combining with a polarizing element such as an analyzer (analyzer), it is possible to know the specific direction of the protein using the fluorescent dye-molecules, and it is possible to capture the dynamic movements individually. In addition, the fine adjustment mechanism of the tilt of the reflecting mirror not only makes it possible to strictly correspond to the focal length of the objective lens used, but also to change the refractive index of the medium surrounding the sample (corresponding to different media, temperature change of the sample). It is possible to correspond closely. The interference fringes of the illumination system caused by the coherence peculiar to the laser beam are averaged in a short time by the rotating reflecting mirror, and as a result, the same effect as removed is obtained. It is possible to obtain excellent effects such as.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an optical system including a rotary annular total reflection illumination mechanism according to the present embodiment.

FIG. 2 is a sectional view of a reflecting mirror tilt fine adjustment mechanism.

3 is a sectional view taken along the line AA in FIG.

FIG. 4 is an explanatory diagram of an optical system in a state in which the tilt of the reflecting mirror is changed in the reflecting mirror tilt fine adjustment mechanism.

FIG. 5 is a principle diagram of total internal reflection illumination using an evanescent field.

FIG. 6 is an optical system diagram of a conventional central shading ring zone total reflection illumination.

FIG. 7 is a diagram showing a focused state of an object plane using Koehler illumination.

[Explanation of symbols]

51 laser fiber light source 52 Collector lens 53 Front focal plane of convex lens 56 54 motor 55 Reflector 56 convex lens 57 dichroic mirror 58 Objective lens front focal plane 59 Objective lens 60 Object plane 61 Reflective body 62 Mirror holding hardware 63 Main body hardware 64 Fine adjustment screw 65 lock nuts 66 pivot screw

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Claims (5)

[Claims] 1. A Koehler-type illumination system for illuminating an object by introducing a laser beam to a peripheral portion of a microscope objective lens, wherein the illumination direction by the laser beam can be rotated while being inclined around the axis of the microscope objective lens. A rotating ring total reflection illumination mechanism characterized by the above. 2. The rotary annular total reflection according to claim 1, wherein the illumination system is an illumination system that introduces a laser beam into a peripheral portion of a microscope objective lens and totally reflects it to emit fluorescence. Lighting mechanism. 3. A reflecting mirror equipped with a mechanism capable of finely adjusting the tilt of the reflecting mirror in order to rotate the illumination direction by the laser beam and give an optimum total reflection angle to the objective lens used, and the rotating mirror. The rotary annular total reflection illuminator according to claim 1 or 2, further comprising a rotating means for rotating the rotary annular total reflection illuminator. 4. The rotary annular total reflection illuminator according to claim 3, wherein the rotation center of the rotating means is arranged at a position slightly apart from the rotation center axis of the reflecting mirror. 5. The rotating means including the reflecting mirror has a structure in which the shape and weight are symmetrical with respect to the central axis of rotation so that the vibration due to the rotation of the reflecting mirror does not adversely affect the microscope. The rotary annular total reflection illumination device according to claim 3 or 4.