JPH1090608A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device capable of simultaneously converging and emitting irradiation light on plural parts different from each other on a specimen. SOLUTION: The irradiation light A and B outputted from an irradiating light source 10, branched by a branching optical system 20 and emitted through a condenser lens 31, dichroic mirrors 35 and 41 and the objective lens 42, irradiate simultaneously two points of the specimen 50 existing in a view area of an objective lens 42. Exciting light C outputted from an exciting light source 32 irradiates the specimen 50 through the condenser lens 33 and a band-pass filter, etc. The illumination light D outputted from an illumination light source 45 irradiates the specimen 50 by the condenser lens 46. Fluorescent E and transmission light F generated from the specimen 50 are picked up by a TV camera 62 through the objective lens 42, the dichroic mirror 41, the band-pass filter 44 and a reflection mirror 61 and displayed on a monitor part 64.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope apparatus that irradiates a sample with irradiation light under a microscope to perform, for example, decomposition of a caged reagent, cell destruction, optical tweezers, and recovery from fluorescence fading.

[0002]

2. Description of the Related Art Conventionally, in addition to irradiating a sample with irradiation light under a microscope and observing scattered light and fluorescence generated from the sample, irradiation of the sample with irradiation light allows the caged reagent to emit light. Disassembly, destruction of a sample, movement of a part of a sample as optical tweezers, or recovery of fluorescent fading are performed. This conventional microscope is used for observation of a sample when it is desired to irradiate an irradiation light to a region of μm level such as one cell among a large number of cells as a sample or a local area within one cell. Irradiation light is condensed and irradiated on the sample surface through an objective lens. Here, the irradiation light is applied to only one point on the sample in one irradiation time.

[0003] Therefore, when it is desired to irradiate different parts of a cell with irradiation light, or to irradiate different cells of a living tissue with irradiation light,
Adjustment of the optical system and movement of the sample are performed for each irradiation of the irradiation light. Further, in combination with a scanning device, scanning with irradiation light is performed sequentially or randomly.

[0004]

However, the above-mentioned prior art irradiates only one point on the sample with irradiation light at a certain time, and simultaneously irradiates a plurality of parts on the sample with irradiation light. It does not do. That is, when irradiating the irradiation light to each of the plurality of portions on the sample, the irradiation light is irradiated to each of the plurality of portions at different times. Therefore, for example, the amount of molecules present in two different cells cannot be simultaneously increased using a caged reagent, and two cells separated from each other cannot be simultaneously destroyed. Cannot be moved at the same time,
It is not possible to simultaneously recover the fluorescent fading of a plurality of sites.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problem, and has as its object to provide a microscope apparatus capable of simultaneously irradiating a plurality of different portions on a sample with irradiation light. I do.

[0006]

According to the present invention, there is provided a microscope apparatus comprising: (1) an irradiation light source for outputting a light beam to be irradiated on a sample; and (2) a light beam output from the irradiation light source. 1
A branching optical system that outputs irradiation light and second irradiation light,
(3) an objective lens which receives each of the first and second irradiation lights and focuses and irradiates each of the two points on the sample within the visual field region; and (4) detects a light image from the sample incident on the objective lens. And a detection optical system. According to this microscope, the first and second irradiation light beams to be irradiated on the sample are output by the light beam output from the irradiation light source being split by the splitting optical system and input to the objective lens, and are input to the objective lens. The two points on the sample are focused and irradiated. The light image from the sample incident on the objective lens is detected by the detection optical system.

[0007] An excitation light source for outputting excitation light to be irradiated on the sample, and an excitation optical system for irradiating the sample with excitation light are further provided. The detection optical system may detect fluorescence generated from the sample. Good. In this case, the excitation light output from the excitation light source passes through the excitation optical system and irradiates the sample irradiated with the first and second irradiation lights. It is detected by the detection optical system.

An illumination light source for outputting illumination light to be irradiated on the sample, and an illumination optical system for irradiating the sample with illumination light are further provided. The detection optical system is configured to transmit light generated by irradiating the sample with the illumination light. Light or scattered light may be detected. In this case, the illumination light output from the illumination light source passes through the illumination optical system and irradiates the sample irradiated with the first and second irradiation light, and the transmitted light or the light generated from the sample accompanying the illumination light. The reflected light is detected by the detection optical system.

A first irradiation position adjusting means for adjusting the irradiation position of the first irradiation light on the sample may be further provided, and in addition to this, the irradiation position of the second irradiation light on the sample is adjusted. A second irradiation position adjusting means may be further provided. In this case, since the irradiation positions of the first and second irradiation lights on the sample are adjusted by the first and second irradiation position adjusting means, respectively, the first and second irradiation lights are respectively adjusted on the sample. At a desired position.

The apparatus may further include first irradiation intensity adjusting means for adjusting the intensity of the first irradiation light applied to the sample,
In addition to this, a second irradiation intensity adjusting means for adjusting the intensity of the second irradiation light irradiated on the sample may be further provided.
In this case, since the respective intensities of the first and second irradiation light applied to the sample are adjusted by the first and second irradiation intensity adjusting means, respectively, the first and second irradiation light are respectively applied to the sample. At a desired intensity.

A first irradiation control means for controlling passage and blocking of the first irradiation light may be further provided, and in addition to this, a second irradiation control means for controlling passage and blocking of the second irradiation light.
May be further provided. In this case, the passage and blocking of the first and second irradiation lights are respectively the first and second irradiation lights.
And the second irradiation control means, respectively, so that the sample can be irradiated only with the first and second irradiation light only when necessary.

[0012] An irradiation intensity measuring means for measuring the intensity of both or any one of the first and second irradiation lights may be further provided. In this case, the intensity of each of the first and second irradiation light irradiating the sample is measured and monitored by the irradiation intensity measuring means.

[0013]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description. FIG. 1 is a configuration diagram of a microscope device according to the present invention.

The irradiation light source 10 emits irradiation light to be irradiated on the sample 50. For example, a laser light source such as an Ar ion laser light source or a Nd: YAG laser light source is preferably used. The branching optical system 20 includes the irradiation light source 1
The irradiation light emitted from 0 is input, and is branched into irradiation light A and irradiation light B. The specific configuration of the branch optical system 20 will be described later. The irradiation lights A and B emitted from the branch optical system 20 enter the microscope main body through the condenser lens 31 and pass through the dichroic mirror 35,
The light is reflected by the dichroic mirror 41 and enters the objective lens 42. The objective lens 42 condenses and irradiates the irradiation light A and the irradiation light B to each of two points in the visual field region of the sample 50 placed on the sample stage 43.

The irradiation lights A and B simultaneously irradiating two points of the sample 50 in the visual field region of the objective lens 42 simultaneously photolyze the two points of the caged reagent or destroy two points in the sample. Alternatively, two points of the sample may be simultaneously moved as optical tweezers, or two points of fluorescence fading may be simultaneously recovered. Further, each of the irradiation lights A and B may be pulse light or continuous light depending on the purpose.

On the other hand, the excitation light source 32 emits excitation light C for exciting the fluorescent substance contained in the sample 50, and a laser light source, a xenon lamp, a mercury lamp or the like is preferably used. The excitation light C emitted from the excitation light source 32 is applied to the sample 50 via an excitation optical system. That is, the excitation light C is condensed by the condenser lens 33, only the excitation wavelength component is transmitted by the band-pass filter 34, thereafter, enters the microscope, is reflected by the dichroic mirror 35, and is reflected by the dichroic mirror 4.
1 and is incident on the objective lens 42. Then, the objective lens 42 collects and irradiates the excitation light C onto the visual field region of the sample 50 placed on the sample stage 43, and excites a fluorescent substance contained in the sample 50 to generate fluorescence E. The illumination light source 45 is provided with illumination light D to be irradiated on the sample 50.
And a condenser lens (illumination optical system) 46
Collects and irradiates the illumination light D onto the sample 50.

The fluorescence E generated by irradiating the sample 50 with the excitation light C and the transmitted light F transmitted by irradiating the sample 50 with the illumination light D are detected by the detection optical system. That is, each of the fluorescence E and the transmitted light F enters the objective lens 42, passes through the dichroic mirror 41, passes only a predetermined wavelength component by the bandpass filter 44, is reflected by the reflecting mirror 61, and is reflected by the television camera 6
An image is formed on the imaging surface 2 and is imaged by the television camera 62. The image of the fluorescent light E or the transmitted light F captured by the television camera 62 passes through the image processing unit 63 and the monitor unit 6.
4 and image processing (for example, pseudo colorization of the intensity distribution of the fluorescence E, transmission light F
Is displayed on the monitor unit 64 after the light image is emphasized, and various analysis (for example, calculation of a time change of the intensity of the fluorescence E generated at a certain point on the sample 50) is performed by the data analysis unit 65. Done.

As described above, the dichroic mirror 35
Is for transmitting the irradiation lights A and B and reflecting the excitation light C. The dichroic mirror 41 receives the irradiation light A
And B and the excitation light C are reflected, and the fluorescence E and the transmitted light F are transmitted. Also, the objective lens 42
Respectively, collectively irradiate the irradiation light A and B to each of two points in the field of view of the sample 50, irradiate the excitation light C onto the field of view of the sample 50 and irradiate the fluorescence E generated from the field of view of the sample 50. And the transmitted light F are input and guided to the television camera 62.

According to this microscope apparatus, the irradiation light source 10
Irradiating light A and B emitted from the
The excitation light C emitted from the excitation light source 32 and the illumination light D emitted from the illumination light source 45
0, and each of the fluorescence E and the transmitted light F generated from the sample 50 passes through the detection optical system and is transmitted to the television camera 6.
2 and is displayed by the monitor unit 64.

Therefore, for example, the sample 50 is irradiated with the illumination light D emitted from the illumination light source 45 without emitting the excitation light C from the excitation light source 32, and the branch optical system 20 emitted from the illumination light source 10 is emitted. When the irradiation light A and the light B respectively branched by the above are irradiated on each of two points on the sample 50, the monitor unit 64 displays the irradiation light A and the light B while observing the sample 50 with the transmitted light F from the sample 50. The irradiation position on each sample 50 can be confirmed and adjusted.

The illumination light D emitted from the illumination light source 45 is emitted to the sample 50 without emitting the illumination light A and B from the illumination light source 10, and the excitation light C emitted from the excitation light source 32 is emitted. Is irradiated on the sample 50, the display position of the fluorescence E on the sample 50 can be confirmed by the display of the monitor unit 64 while observing the sample 50 with the transmitted light F from the sample 50. Therefore, even when the sample 50 moves during observation like a biological sample, it is possible to accurately confirm the generation position and intensity distribution of the fluorescence E in the sample 50.

Next, a preferred example of the branch optical system 20 of the microscope apparatus according to the present invention will be described. FIG. 2 is a configuration diagram of an example of the branch optical system.

The irradiation light emitted from the irradiation light source 10 is:
The light enters the branch optical system 20 and is first split into two by the half mirror 21. One of the two split light beams A is reflected by the reflected light beam 22A, passes through the shutter 23A, passes through the neutral density filter 24A at a predetermined transmittance, and enters the half mirror 26. The other irradiation light B is reflected light 2
2B, passes through a shutter 23B, passes through a neutral density filter 24B at a predetermined transmittance, and
Incident on. Then, each of the irradiation lights A and B incident on the half mirror 26 is partially reflected, and the rest is transmitted. The irradiation light A and the irradiation light B reflected by the half mirror 26 are respectively emitted from the branch optical system 20, enter the condenser lens 31, and further enter the microscope body.

Here, a reflecting mirror (irradiation position adjusting means) 22
The inclination of each of A and 22B can be set variably, and the position where each of the irradiation lights A and B is irradiated onto the sample 50 can be adjusted according to the inclination. When the shutters (irradiation control means) 23A and 23B are open, the illumination light A
And B are passed through and irradiated onto the sample 50. When the sample is closed, the irradiation lights A and B are blocked and the sample 50 is not irradiated. If these shutters 23A and 23B are opened and closed at the same timing, the irradiation lights A and B can be irradiated onto the sample 50 at the same time.
If necessary, the sample can be irradiated with the irradiation light beams A and B on the sample 50 by shifting the opening and closing by a required time, if necessary. Further, each of the neutral density filters (irradiation intensity adjusting means) 24A and 24B
And B, the intensity of irradiation on each sample 50 is adjusted.

The irradiation light A reflected by the half mirror 26 and the irradiation light B transmitted therethrough are respectively transmitted to the branch optical system 20.
And a photometer (irradiation intensity measuring means) 28
And the amount of light is measured, and from the measured value, the sample 50
The intensity of each of the irradiation light A and the irradiation light B applied to the light source can be obtained.

Next, another preferred example of the branch optical system 20 of the microscope apparatus according to the present invention will be described. FIG. 3 is a configuration diagram of another example of the branch optical system.

The irradiation light emitted from the irradiation light source 10 is:
After entering the splitting optical system 20, first, the polarization plane is rotated by the half-wave plate 29, and the light is split into two by the half mirror 21. One of the two split irradiation lights A is reflected light 22A.
, And the polarization plane is rotated by the half-wave plate 25A through the shutter 23A, and is incident on the polarization beam splitter 27. The other irradiation light B is reflected light 22
The light is reflected by B, passes through a shutter 23B, is rotated by a half-wave plate 25B, and is incident on a polarization beam splitter 27.

The polarization beam splitter 27 reflects or transmits light according to the polarization direction of incident light. For example, a polarization beam splitter using a dielectric polarizing film, a Glan laser prism using a crystal, or the like is used. Used. Therefore, the irradiation light A incident on the polarization beam splitter 27
Each of B and B is partially reflected and the rest is transmitted. That is, the first polarization component of the irradiation light A transmitted through the polarization beam splitter 27 and the second polarization component of the irradiation light B reflected therefrom.
Are emitted from the branching optical system 20, and
The light enters the condenser lens 31 and further enters the microscope body. In addition, one of the first and second polarization components represents a p-polarization component, and the other represents an s-polarization component.

Here, the reflecting mirror (irradiation position adjusting means) 22
A and 22B and shutter (irradiation control means) 23
Each of A and 23B is the same as that described in FIG. The half-wave plates (irradiation intensity adjusting means) 25A and 25B respectively adjust the intensity of each of the irradiation light A and B irradiated onto the sample 50 in the same manner as each of the neutral density filters 24A and 24B in FIG. Is what you do. That is, depending on the polarization state of the irradiation light A emitted from the half-wave plate 25A, the ratio of transmission of the irradiation light A through the polarization beam splitter 27 is different.
The proportion reflected by the polarization beam splitter 27 differs depending on the polarization state of the irradiation light B emitted from 5B. As described above, when adjusting the intensity of each of the irradiation lights A and B applied to the sample 50 using the half-wave plates 25A and 25B, the loss of each of the irradiation lights A and B in the polarization beam splitter 27 is reduced. Can be suppressed, and the irradiation light A and B can be efficiently irradiated onto the sample 50.

The second polarized light component of the illuminating light A reflected by the polarizing beam splitter 27 and the first polarized light component of the transmitted illuminating light B are respectively emitted from the branching optical system 20 and subjected to a photometer (irradiation intensity measurement). (Means) The amount of light incident on the sample 50 is measured, and the intensity of each of the irradiation light A and the irradiation light B applied to the sample 50 can be obtained from the measured value.

Next, an experimental example using the microscope apparatus according to the present invention will be described. Here, each of the irradiation lights A and B acts as decomposition light for photodecomposing the caged reagent.

The experimental system and experimental conditions are as follows. As an irradiation light source 10, an Ar ion laser light source (wavelength 351 nm, continuous oscillation, electromagnetic shutter 1/16 second),
Alternatively, an Nd: YAG laser light source (wavelength: 355 nm, pulse oscillation, pulse width: 5 ns) was used. Branch optical system 20
The structure shown in FIG.
The light beam emitted from the light source is branched by the branching optical system 20 to obtain irradiation lights A and B. The objective lens 42 used had a magnification of 100 times. The sample 50 is a cultured cell (NG108-15). A caged reagent that decomposes and releases Ca2 + ions upon irradiation with each of the irradiation lights A and B is added to the sample 50. caged Ca2 + (o-nitrophenyl-EGTA / AM) ) And flu as a fluorescent reagent for detecting Ca2 + ions.
o-3 / AM was introduced.

Irradiation Light A and B Irradiated on Sample 50
Each spot was imaged by a television camera 62 via a detection optical system including an objective lens 42 and the like, and observed by a monitor 64. The irradiation diameter of each of the irradiation lights A and B was about 1 μm. Further, by adjusting the inclination of each of the reflecting mirrors 22A and 22B, the irradiation lights A and B are adjusted.
It was confirmed that the irradiation position on each sample 50 could be adjusted.

After simultaneously irradiating the irradiation light A and the irradiation light B to each of two different cells, the excitation light source 32
The sample 50 is irradiated with the excitation light C emitted from the sample, and the fluorescence E generated from the fluorescent reagent is imaged by the television camera 62 via the detection optical system including the objective lens 42 and the like, and observed by the monitor unit 64. In each of the two cells irradiated with A and B, the caged reagent was photolyzed.
Ca2 + is increased, but Ca2 + is
Was not increased. Further, after that, when the cells were irradiated with the excitation light C and the fluorescence E was continuously observed, the intensity of the fluorescence E from the two cells irradiated with the irradiation light A and B was gradually decreased, and the intensity of the fluorescence E from the other cells was decreased. The fluorescence E slightly increased. This is considered that Ca2 + increased in the two cells due to the irradiation with the irradiation light A and B diffused into the other cells.

After irradiating the irradiation light A and the irradiation light B simultaneously to each of two points in a single cell, the excitation light source 3
The sample 50 is irradiated with the excitation light C emitted from the sample 2, the fluorescence E generated from the fluorescent reagent is imaged by the television camera 62 via the detection optical system including the objective lens 42 and the like, and the monitor unit 64
As a result, the caged reagent was photolyzed and Ca2 + increased at each of the two points in the cells irradiated with the irradiation lights A and B.
It was confirmed that a2 + did not increase. After that, the cells were irradiated with the excitation light C and the fluorescence E was continuously observed.
The intensity of the fluorescence E from the point gradually decreased, the fluorescence intensity gradually increased around the point, and the generation of the fluorescence E from the cells tended to be uniformed over time. This is considered to be due to the fact that Ca2 + locally increased by irradiation with the irradiation lights A and B diffuses into the cells.

Further, the illumination light D from the illumination light source 45 is applied to the sample 50, and the optical image of the transmitted light F is captured by the television camera 62 and displayed on the monitor section 64 to confirm the shape of the cells. At the same time, the position of fluorescence emission in the cells could be confirmed.

The present invention is not limited to the above embodiment, and various modifications and applications are possible. For example, FIG.
In the embodiment shown in (1), the transmission type microscope observes the transmitted light F from the sample 50 by irradiating the sample 50 with the illumination light D, but may be a reflection type microscope observing the reflected light from the sample 50. .

In the above experimental example, the irradiation lights A and B were used as the decomposition light for photodecomposing the caged reagent introduced into the sample 50. However, the inside of the sample was destroyed or the sample was used as optical tweezers. The microscope apparatus according to the present invention is also effective when a part is moved or when fluorescence fading is recovered. The wavelengths and intensities of the irradiation lights A and B and the continuous output or the pulse output are optimally set according to the purpose.

[0039]

As described above in detail, according to the present invention, the first and second irradiation light beams to be irradiated on the sample are output by the light beam output from the irradiation light source being split by the splitting optical system. Then, the light is input to the objective lens, and is condensed and irradiated on each of two points on the sample in the field of view. On the other hand, a light image from the sample incident on the objective lens is detected by the detection optical system. With such a configuration, it is possible to simultaneously irradiate the irradiation light to two points in the field of view of the objective lens.
It is possible to apply a light stimulus to the two points at the same time and analyze the subsequent reaction.

The excitation light output from the excitation light source passes through the excitation optical system and irradiates the sample irradiated with the first and second irradiation lights. It is detected by the detection optical system. With such a configuration, it is possible to detect the fluorescence generated from the position on the sample irradiated with the first and second irradiation lights and the surrounding area.

The illumination light output from the illumination light source passes through the illumination optical system and irradiates the sample irradiated with the first and second irradiation lights, thereby transmitting the transmitted light or the light generated from the sample. The reflected light is detected by the detection optical system.
With such a configuration, it is possible to irradiate the first and second irradiation lights to a desired position in the sample while observing the sample with the transmitted light image, and to determine the fluorescence generation distribution in the sample. Can be analyzed.

Therefore, for example, a caged reagent is introduced into a cell group constituting a network, such as a nerve cell, and two of the cells are simultaneously activated by irradiating irradiation light, thereby simultaneously activating the two cells. The reaction of other cells can be detected, and it is possible to analyze the difference in response between the case where two such cells are activated simultaneously and the case where only one cell is activated. In general, the activity of cells in a living body is often controlled by the activities of a plurality of cells interacting with each other. Thus, the present invention is effective in analyzing information transmission between a plurality of cells and the like. It is.

Also, depending on the magnification of the objective lens, the luminous flux diameter of the irradiation light can be made smaller than the size of the cell, so that two different points in one cell are irradiated with the irradiation light at the same time. Stimulation can be performed. For example,
Due to the spatial disparity in the arrangement, movement path, or activation state of molecules within one cell, the degree and direction of expression of cell functions (eg, the direction in which protrusions are extended, the direction in which information molecules are released) are changed. According to the present invention, two different points (e.g., near the cell membrane and in the nucleus) in the cell into which the caged reagent has been introduced are activated and irradiated simultaneously with irradiation light. , The function of the cell can be analyzed.

Further, according to the present invention, the irradiation positions of the first and second irradiation lights on the sample are respectively the first and second irradiation light.
Are adjusted by the respective irradiation position adjusting means, so that each of the first and second irradiation light can be applied to a desired position on the sample. Further, since the respective intensities of the first and second irradiation light applied to the sample are adjusted by the first and second irradiation intensity adjusting means, respectively, the first and second irradiation light can be respectively applied to the sample by a desired amount. Irradiation at high intensity is possible. Further, since the passage and blocking of the first and second irradiation lights are controlled by the first and second irradiation control means, respectively, the first and second irradiation lights are irradiated onto the sample only when necessary. I can do it. In addition, the intensity of each of the first and second irradiation light applied to the sample is measured and monitored by the irradiation intensity measuring means.
With such a configuration, each of the first and second irradiation lights can be applied to any two points on the sample at an arbitrary intensity and at an arbitrary time, so that the microscope according to the present invention can be used. The device is extremely effective in elucidating the various functions of the cells described above.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a microscope device according to the present invention.

FIG. 2 is a configuration diagram of an example of a branch optical system.

FIG. 3 is a configuration diagram of another example of the branch optical system.

[Explanation of symbols]

Reference Signs List 10 ... irradiation light source, 20 ... branch optical system, 21 ... half mirror, 22A, 22B ... reflection mirror, 23A, 23B ... shutter, 24A, 24B ... darkening filter, 25A, 25B
... 1/2 wavelength plate, 26 half mirror, 27 polarization beam splitter, 28 photometer, 29 1/2 wavelength plate, 31 condenser lens, 32 excitation light source, 33 condenser lens, 34 … Band pass filter, 35… dichroic mirror, 41… dichroic mirror, 42… objective lens, 43… sample stage, 44… band pass filter, 45… transmission light source, 46… condenser lens, 50… sample, 61… reflection Mirror, 62: TV camera, 63: Image processing unit, 64: Monitor unit, 65: Data analysis unit, A, B ...
Irradiation light, C: excitation light, D: illumination light, E: fluorescence, F: transmitted light.

Claims (10)

[Claims] 1. An irradiation light source for outputting a light beam to be irradiated on a sample, and a branch optical system for splitting a light beam output from the irradiation light source and outputting a first irradiation light and a second irradiation light. An objective lens that receives each of the first and second irradiation lights and collects and irradiates each of the two points on the sample within a visual field region; and detects a light image from the sample that has entered the objective lens. A microscope apparatus comprising: a detection optical system; 2. The apparatus according to claim 1, further comprising: an excitation light source for outputting excitation light to be irradiated on the sample, and an excitation optical system for irradiating the sample with the excitation light, wherein the detection optical system includes a fluorescent light generated from the sample. The microscope apparatus according to claim 1, wherein: 3. The apparatus according to claim 1, further comprising: an illumination light source that outputs illumination light to be irradiated on the sample; and an illumination optical system that irradiates the sample with the illumination light. The microscope apparatus according to claim 1, wherein transmitted light or scattered light generated by irradiating the microscope is detected. 4. The microscope apparatus according to claim 1, further comprising first irradiation position adjusting means for adjusting the irradiation position of the first irradiation light on the sample. 5. The microscope apparatus according to claim 4, further comprising second irradiation position adjusting means for adjusting the irradiation position of the second irradiation light on the sample. 6. The microscope apparatus according to claim 1, further comprising first irradiation intensity adjusting means for adjusting the intensity of the first irradiation light applied to the sample. 7. The microscope apparatus according to claim 6, further comprising a second irradiation intensity adjusting unit that adjusts the intensity of the second irradiation light applied to the sample. 8. The microscope apparatus according to claim 1, further comprising first irradiation control means for controlling passage and blocking of the first irradiation light. 9. The microscope apparatus according to claim 8, further comprising second irradiation control means for controlling passage and blocking of the second irradiation light. 10. The microscope apparatus according to claim 1, further comprising an irradiation intensity measuring unit for measuring an intensity of both or any one of the first and second irradiation lights.