JPH08254654A - Confocal point polarization microscope - Google Patents

Abstract

PURPOSE: To provide a device which is provided with a means capable of fixing fluorescence dyestuff on the specified position of a sample, and obtaining the polarization degree of the fluorescence dyestuff in a state where it has depth resolution in an optical microscope. CONSTITUTION: Single color light emitted from a laser light source 1 is guided to an objective lens 4 by a dichroic mirror 7, and is focused on the sample 6 on a sample plate. The light from a fluorescence image on a focusing point is transmitted through the lens 4 again, passes through a filter 8 suitable for the characteristic of the fluorescence dyestuff, then is branched into two optical paths by the dichroic mirror 9. The branched light beams are guided to polarizing boards 10 and 13 crossing at a right angle with each other, so that two images in accordance with the polarization of the fluorescence dyestuff can be obtained and the polarizing direction of the fluorescence dyestuff can be estimated by combining and analyzing the two images.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope for observing a cross section of a sample containing a fluorescent dye.

[0002]

2. Description of the Related Art The optical microscope technique is one of the main techniques for directly observing information such as temporal changes of constituent elements in cells in a living state of a biological sample. Recent advances in optical microscopy have succeeded in extending the resolution limit of the observation method that depends on the refractive index of the target sample to the sub-nanometer range by utilizing the luminescence phenomenon of fluorescent substances. It is becoming possible to observe the behavior of a single protein molecule, which could only be observed with an electron microscope, in a living state without going through the process of sample fixation.

Since the sample can be observed in a living state, changes over time in specific elements of the sample, such as changes in pH and calcium ion concentration, are replaced with changes in the fluorescence intensity of the fluorescent substance. Therefore, it is becoming possible to extract information having both temporal resolution and spatial resolution. In addition, by binding a fluorescent dye to a specific protein and observing the polarization of the fluorescent dye with two polarizing plates orthogonal to each other, information on the structural change of the functional protein such as orientation and rotation of the protein can be directly obtained. be able to. Regarding these technologies, Kazuhiko Kinoshita et al., Journal of Cell Bio
logy 115: 67-71 (1991).

[0004]

In the above-mentioned conventional optical microscope technique, although the resolution in the observation plane direction is sufficiently obtained, the resolution in the depth direction is not sufficiently obtained. Therefore, for example, a specific cytoplasm in a cell is obtained. If you try to observe the behavior of the, you will get the information of the depth position other than the plane in the depth direction including the cytoplasm you want to observe as noise, and it will be difficult to identify the protein of the obtained information source. There were cases.

Further, the conventional confocal microscope has a means for acquiring a fluorescence image of the cross section of the sample, but has no means for measuring a minute structural change such as local rotation of the sample of the cross section.

An object of the present invention is to provide an apparatus having means for observing a fine structural change such as rotation of a specific portion of a sample in an optical microscope.

[0007]

In order to solve the above-mentioned problems, a fluorescent dye is immobilized on a specific site of a sample, and a means for acquiring the polarization degree of the fluorescent dye in a form having depth resolution is provided. In the optical path from the objective lens of the microscope to the observation light receiving surface through the confocal pinhole by applying the focusing microscope technology, the optical path is divided into two parts before and after the pinhole, and the two branched optical paths are orthogonal to each other. It is only necessary to introduce two polarizing plates, observe the images that have passed through the respective polarizing plates, and compare the two images to observe the degree of polarization of the fluorescent dye and the change in the degree of polarization.

[0008]

[Function] In the confocal microscope, the confocal optical system places a pinhole at a position optically conjugate with the focus position of the objective lens with respect to the sample, so that light from other than the focus position is completely eliminated, and An optical slice image of a sample can be acquired without destroying the sample. Therefore, by dividing the optical path into two in the front stage or the rear stage of the pinhole and passing the two polarizing plates orthogonal to each other, the direction of each polarizing plate in the specific slice area of the fluorescent dye fixed to the sample is changed. The degree of polarization can be detected respectively. By comparing the two polarization images thus detected, it is possible to obtain information on the tilt, movement, rotation, etc. of the sample.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a schematic diagram of the apparatus configuration of one embodiment of the present invention. The monochromatic light emitted from the laser light source 1 passes through the lens 201, the pinhole 202, and the lens 203 to become parallel light having the same size as the pupil of the objective lens 4 and reaches the neutral density filter 3. Next, the light passing through the neutral density filter 3 is guided to the objective lens 4 by the dichroic mirror 7. At this time, the position of the dichroic mirror can be freely moved in the XY direction by the control analysis device, and the image forming point of the objective lens can be moved to an arbitrary point on the XY plane of the sample.

The light that has passed through the objective lens 4 is focused by using a tomographic plane in an arbitrary Z-axis direction of the sample 6 on the sample plate 5 whose position can be changed in the Z-axis direction as a focal plane. The in-focus fluorescence image passes through the objective lens 4, the dichroic mirror 7, and then the filter 8 that matches the characteristics of the fluorescent dye, and is then branched into two optical paths by the dichroic mirror 9.

The branched light is guided to two polarizing plates 10 and 13 which are orthogonal to each other. Polarizing plates 10, 1
The fluorescent image that has passed through 3 is formed by the lenses 111 and 141, and only the same light as the focal point of the objective lens can pass through the pinholes 112 and 142. The passed light passes through the lenses 113 and 143 again, and the detection device 1
It is guided to 2,15. The information obtained by the detection devices 12 and 15 is stored in the control analysis device 16 and is moved by moving the position of the dichroic mirror 4 or the height of the sample plate 5 by scanning on the XY plane, the XZ plane, or the like. It is possible to reconstruct the measurement result of the sample tomographic plane and estimate the polarization direction of the fluorescent dye of the sample.

FIG. 2 shows a slice image of the sample observed by the apparatus of the above embodiment.

In the tomographic image 17 of the sample that does not pass through the polarizing plate, all the fluorescent dyes lined up in the cross section 18 of the sample are observed, but by passing through the polarizing plates 10 and 13, an image 20 corresponding to the polarization of the fluorescent dye is obtained. , 21 can be obtained, and the polarization direction of the fluorescent dye can be estimated by analyzing them in combination.

It is desirable to use parallel light for inserting the polarizing plate and branching the optical path by the dichroic mirror, and it is desirable that the latter stage of the objective lens 4 is parallel light. At this time, it is desirable that the polarizing plate is inserted at the position of the parallel light in the front stage of the lenses 111 and 141 or the position after the parallel light is processed in the rear stage of the lenses 113 and 143.

Further, in the present embodiment, the light is branched into two optical paths and each branched light is introduced into the polarizing plate. However, by using a means for rotating the polarizing plate, information on the directions of polarization planes different in one optical path is obtained. The same effect can be obtained by acquiring twice and comparing the information. However, in this case, it is necessary to consider the fading of the fluorescent dye due to light irradiation, and to estimate the fading amount when the control analysis device 16 evaluates the result.

[0016]

EFFECTS OF THE INVENTION Since the present invention is configured as described above, in an optical microscope, a fluorescent dye is immobilized on a specific site of a sample, and the degree of polarization of the fluorescent dye has a depth resolution. There is an effect that can be acquired.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a device configuration of an embodiment of the present invention.

FIG. 2 is a diagram showing an example of a fluorescence observation image using an embodiment of the present invention.

[Explanation of symbols]

1 ... Laser light source, 201 ... Lens, 202 ... Pinhole, 203 ... Lens, 3 ... Filter, 4 ... Objective lens, 5 ... Sample plate, 6 ... Sample, 7 ... Dichroic mirror, 8 ... Filter, 9 ... Dichroic mirror,
10 ... Polarizing plate, 111 ... Lens, 112 ... Pinhole,
113 ... Lens, 12 ... Detection device, 13 ... Polarizing plate, 14
1 ... Lens, 142 ... Pinhole, 143 ... Lens, 1
5 ... Detection device, 16 ... Control analysis device, 17 ... Sample slice image, 18 ... Sample cross section, 19 ... Fluorescent dye orientation direction, 2
0 ... Sample slice image after passing through the polarizing plate, 21 ... Sample slice image after passing through the polarizing plate.

Claims (2)

[Claims] 1. In an optical microscope, a light source, a means for branching a plurality of optical paths, a means for measuring polarized light in a plurality of directions arranged at a subsequent stage, and only light having the same focal point as that of an objective lens. A confocal polarization microscope characterized by having a means for passing it. 2. In an optical microscope, a laser light source, a dichroic mirror that guides laser light to an objective lens and that is variable in the XY directions, an objective lens, a sample plate that is variable in the Z axis direction, and a rear stage of the objective lens. The fluorescent filter, the dichroic mirror that splits the optical path into two, the two polarizing plates with different polarization plane orientations that are placed in the subsequent stage, and only the light with the same focal point as that of the objective lens passes A lens and a pinhole to be operated, a detector for detecting the intensity of light in the subsequent stage, a position of the dichroic mirror variable in the X-Y direction and a position of the sample plate variable in the Z-axis direction, and the respective positions are controlled. The confocal image recording device according to claim 1, further comprising: a control analysis unit having a function of recording the light intensity data of the detection unit and reconstructing it as image data after scanning the focal position. Polarizing microscope.