JPH09329749A - Optical scanning type microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical scanning type microscope constituted so that an image with excellent contrast is obtained by minimizing the discoloring of fluorescence in a sample caused by the irradiation of a laser light beam when laser power and a photodetector are adjusted before image data is obtained. SOLUTION: The optical scanning type microscope 1 is provided with a laser light source 101 emitting the laser light beam and scanning mirrors 107 and 108 two-dimensionally scanning the sample 105 with the laser light beam. Besides, it is provided with a laser irradiation control mask 10 arranged in an optical path to the sample 105 from the light source 101 and set for intermittently irradiating the sample 105 with the laser light beam moved in a horizontal direction by the horizontal scanning action of the mirrors 107 and 108.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an optical scanning microscope.

[0002]

2. Description of the Related Art An optical scanning microscope, for example, a laser scanning microscope is known as a microscope for irradiating a biological sample with light and observing a phenomenon occurring in the biological sample.

FIG. 5 is a block diagram of a confocal laser scanning microscope.

The confocal laser scanning microscope 100 includes a laser light source 101 that emits excitation laser light and an excitation filter 10.
2, a beam expander 104 that expands the excitation laser light to a size that fills the pupil plane of the objective lens 103, a dichroic mirror 106 that does not transmit the excitation laser light, but transmits the fluorescence excited by the sample 105, and the laser light. Scanning mirrors 107 and 108 for two-dimensionally scanning a beam, a condenser lens 109, and an absorption filter 11 for removing laser light.
0, a condenser lens 111, and a pinhole plate 112 which is arranged at a position conjugate with the focal plane of the objective lens 103 and has a pinhole 112a through which only the fluorescence condensed by the condenser lens 111 passes. A fluorescence image system that includes a fluorescence detector 113 that detects fluorescence that has passed through the pinhole 112a and converts it into an electric signal, a monitor 121, a CPU 122, and the like, and that images the sample 105 based on the electric signal from the fluorescence detector 112. And 120.

The operation of the confocal laser scanning microscope having the above configuration will be described.

The excitation laser light emitted from the laser light source 101 is generated by the excitation filter 102 and the beam expander 1.
After passing through 04, it is reflected by the dichroic mirror 106, is two-dimensionally shaken by the scanning mirrors 107 and 108, and is irradiated as a spot on the focal plane on the sample 105 by the condenser lens 109 and the objective lens 103.

The fluorescence of the spot portion excited by the irradiation of the excitation laser light, the objective lens 1 together with the excitation laser light.
03 to condenser lens 109, scanning mirrors 107 and 108
The optical path is reversed to and is separated from the excitation laser light by the dichroic mirror 106.

The fluorescence passing through the dichroic mirror 106 passes through the absorption filter 110, and then the condenser lens 1
The light is focused on the pinhole 112 at 11, and the fluorescence detector 113
Is converted into an electric signal and is displayed as an image by the fluorescence image system 120.

The spot of the excitation laser light is the scanning mirror 10.
7, 108, the image acquisition range (5
(12 × 480 pixels), the fluorescence image system 120 can obtain image data in a corresponding range.

Since only the fluorescence on the focal plane of the sample 105 passes through the pinhole 112a, unnecessary scattered light is removed by the pinhole 112, and the fluorescence image system 120 produces an image with significantly improved resolution and contrast in the depth direction. It is possible to obtain.

6 and 7 are views for explaining a conventional optical scanning method.

By the way, when performing fluorescence observation of a biological sample, when the biological sample receives strong light such as laser light for a long time due to repeated scanning as shown in FIG. 6, fading occurs and the amount of fluorescence changes. It will fall. For example, fluorescence may fade due to laser light irradiation during adjustment of laser power or photodetector adjustment (during image acquisition condition adjustment) performed prior to image data acquisition, and an image with good contrast may not be obtained. There is.

In order to suppress fading when adjusting the image acquisition conditions, the following two methods have been conventionally used.

The first method is a method of intermittently irradiating the sample with laser light (for example, every 1 second) to shorten the fluorescence excitation time for the sample surface.

The second method is to reduce the number of scanning lines per frame from, for example, 480 to 120, perform horizontal scanning at intervals of 1st, 5th and 9th, and then 2 4th scanning so that the scanning lines are between the first scanning lines such as the 6th line, the 6th line, the 10th line, then the 3rd line, the 7th line, the 11th line, and finally the 4th line, the 8th line, and the 12th line. This is a method of scanning the entire screen by means of shortening the excitation time of the excitation light for one spot of the sample 105 (FIG. 7).
reference).

[0016]

However, in the first method, the irradiation of the laser beam is intermittent, but the irradiation range is the entire surface of the sample, so that the sample 1 is adjusted when the image acquisition conditions are adjusted.
No. 05 is irradiated with laser light more than necessary, fluorescence is fading in many parts, and there is a problem that an image obtained after adjustment has extremely poor contrast.

In the second method, the fading area in the vertical scanning direction can be reduced, but since the laser beam is still radiated on all scanning lines during horizontal scanning,
The fading of fluorescence in the horizontal scanning region is the same as that of the conventional one, and there is a problem that an image with good contrast cannot be obtained.

The present invention has been made in view of the above circumstances, and its problem is to detect fluorescence in a sample due to laser light irradiation during adjustment of laser power and photodetector performed prior to acquisition of image data. An object of the present invention is to provide an optical scanning microscope capable of obtaining an image with good contrast while minimizing fading.

[0019]

In order to solve the above-mentioned problems, an optical scanning microscope according to a first aspect of the present invention comprises a light source for emitting a light beam and a scanning for two-dimensionally scanning the light beam on a sample. In the optical scanning microscope including means, for intermittently irradiating the sample with the light beam that is arranged in the optical path from the light source to the sample and moves in the horizontal direction by the horizontal scanning of the scanning means. The light-shielding means is provided.

When the light beam is moved in the horizontal direction by the horizontal scanning of the scanning means, the sample is irradiated by the laser beam because the irradiation of the sample is intermittent due to the light shielding means arranged in the optical path from the light source to the sample. It is possible to minimize the fading of the fluorescence inside.

An optical scanning microscope according to a second aspect of the present invention is the optical scanning microscope according to the first aspect, wherein the light shielding means is provided at a position substantially conjugate with the sample surface.

Since the light shielding means is provided at a position substantially conjugate with the sample surface, the sample is irradiated with an arbitrary amount of excitation laser light by changing the size and shape of the region where the excitation laser light is shielded. be able to.

An optical scanning microscope according to a third aspect of the present invention is the optical scanning microscope according to the first aspect, further comprising drive means for moving the light shielding means for each vertical scanning. Is characterized by.

Since the light-shielding means is moved by the driving means for each vertical scanning, the irradiation of the laser beam is intermittent also in the vertical scanning direction, and the fading of fluorescence in the sample can be made uniform.

[0025]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of a laser scanning microscope according to an embodiment of the present invention, and FIG. 2 is an enlarged view of a laser irradiation control mask. The same parts as those of the laser scanning microscope of FIG. And its description is omitted.

Laser scanning microscope (optical scanning microscope) 1
Is a laser irradiation control mask (shading means) 10 and a drive unit (driving means) for driving the laser irradiation control mask 10.
20 differs from the conventional laser scanning microscope shown in FIG.

The laser irradiation control mask 10 is formed by alternately forming slits (light transmitting portions) 11 for transmitting excitation laser light and light shielding portions 12 on a rectangular flat plate formed of a light shielding material.

Further, the laser irradiation control mask 10 is provided at a position (primary image plane of the objective lens 103) substantially conjugate with the surface of the sample 105, orthogonal to the optical axis. The laser irradiation control mask 10 can move in the direction orthogonal to the optical axis.

The drive unit 20 drives the laser irradiation control mask 10 according to an instruction from the CPU 122.

FIG. 3 is a view for explaining an example of the optical scanning method applied to the optical scanning microscope according to the embodiment of the present invention.

The operation of the laser scanning microscope having the above configuration will be described with reference to FIGS. 1, 2 and 3. The drive unit 2
It is assumed that the laser irradiation control mask 10 has been moved to the optical axis in advance by 0.

The excitation laser light emitted from the laser light source 101 is excited by the excitation filter 102 and the beam expander 1.
After passing through 04, it is reflected by the dichroic mirror 106, is two-dimensionally swung by the scanning mirrors 107 and 108, and is condensed on the laser irradiation control mask 10 by the condenser lens 109.

This laser light is emitted from the laser irradiation control mask 10
The sample 105 is irradiated with light through the slit 11 to excite fluorescence.

This fluorescence is emitted from the objective lens 103 through the laser irradiation control mask 10 and the condenser lens 1 together with the excitation laser light.
09, the optical path goes backward to the scanning mirrors 108 and 107, and is separated from the excitation laser light by the dichroic mirror 106.

The fluorescence passing through the dichroic mirror 106 passes through the absorption filter 110, and then the condenser lens 11
At 1, the light is focused on the pinhole 112a. Then, the fluorescence is received by the fluorescence detector 113, converted into an electric signal, and displayed as an image by the fluorescence image system 120.

During the above operation, the sample 105 is intermittently irradiated with the excitation laser light by the slit 10 of the laser irradiation control mask, so that the irradiation amount of the excitation laser light is greatly reduced.

The CPU 122 uses the laser irradiation control mask 1
The drive unit 20 is controlled so that 0 is moved left and right by the width of the slit 11 for each horizontal scanning. By doing so, the fluorescence emitted from the sample 105 upon being excited by the excitation laser light can be suppressed to the necessary minimum, and the sample 1
It is possible to uniformly irradiate the laser beam on 05 (see FIG. 3).

FIG. 4 is a diagram for explaining another example of the optical scanning method applied to the optical scanning microscope according to the embodiment of the present invention.

The scanning is performed every four frames as in the case of FIG. 7, and the laser irradiation control mask 10 may be moved by the drive unit 20 for each frame to change the region irradiated by the excitation laser light. By doing so, it becomes possible to uniformly irradiate the sample 105 with the laser light as in the case described above.

Furthermore, the irradiation of the laser beam is intermittent (for example, 1
It may be performed every second) to shorten the time of irradiation with the excitation laser light.

Furthermore, the above three methods may be combined appropriately.

Next, an example of fluorescence observation will be described.

Prior to fluorescence observation of cells stained with FITC as the sample 105, the laser power and the photodetector were adjusted (image acquisition condition adjustment) by applying the optical scanning method shown in FIG. After that, when fluorescence observation was performed using the laser scanning microscope 1, the obtained image had fading suppressed to the minimum and the fading in the sample was uniform and had a good contrast.

As dyeing dyes, other than FITC,
BODIPY FL, Rhod
Amine (Rhodamine), Texas Red (Texas Red), Propium Iodide (propidium iodide) and the like can be used.

[0046]

As described above, according to the optical scanning microscope of the invention described in claim 1, when the light beam is moved in the horizontal direction by the horizontal scanning of the scanning means, it is arranged in the optical path from the light source to the sample. Since the irradiation of the sample is intermittent by the light-shielding means provided, the fading of the fluorescence in the sample due to the irradiation of the laser light can be minimized. Therefore,
When the sample is observed by actually irradiating it with the excitation laser light, an image with good contrast can be obtained.

According to the optical scanning microscope of the second aspect of the invention, since the light shielding means is provided at a position substantially conjugate with the sample surface, the size and shape of the region where the excitation laser beam is shielded is changed to change the size of the sample. It is possible to irradiate an arbitrary amount of excitation laser light on the top.

According to the optical scanning microscope of the third aspect of the invention, since the light shielding means is moved by the driving means for each vertical scanning, the irradiation of the laser beam is intermittent also in the vertical scanning direction, and the sample is The fading of the fluorescence inside can be made uniform.

[Brief description of drawings]

FIG. 1 is a block diagram of a laser scanning microscope according to a first embodiment of the present invention.

FIG. 2 is an enlarged view of a laser irradiation control mask.

FIG. 3 is a diagram illustrating an example of an optical scanning method applied to the optical scanning microscope according to the embodiment of the present invention.

FIG. 4 is a diagram illustrating another example of the optical scanning method applied to the optical scanning microscope according to the embodiment of the present invention.

FIG. 5 is a diagram illustrating a conventional optical scanning method.

FIG. 6 is a diagram illustrating a conventional optical scanning method.

FIG. 7 is a diagram illustrating a conventional optical scanning method.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Laser scanning microscope (light scanning microscope) 10 Laser irradiation control mask (light-shielding means) 20 Driving part (driving means) 101 Light source 105 Samples 107, 108 Scanning mirror (scanning means)

Claims (3)

[Claims] 1. A light scanning microscope comprising a light source for emitting a light beam and a scanning means for two-dimensionally scanning the light beam on a sample, the light scanning microscope being disposed in an optical path from the light source to the sample, An optical scanning microscope, comprising: a light-shielding device for intermittently irradiating the sample with the light beam that moves in the horizontal direction by the horizontal scanning of the scanning device. 2. The optical scanning microscope according to claim 1, wherein the light shielding unit is provided at a position substantially conjugate with the sample surface. 3. The optical scanning microscope according to claim 1, further comprising a driving unit that moves the light shielding unit for each vertical scanning.