JP2006003521A5

JP2006003521A5 -

Info

Publication number: JP2006003521A5
Application number: JP2004178253A
Authority: JP
Filing date: 2004-06-16
Publication date: 2007-08-02
Anticipated expiration: 2024-06-16

Claims

A first laser light source for generating observation excitation laser light having a one-photon excitation wavelength;
A second laser light source that generates excitation laser light for observation of a multiphoton excitation wavelength;
A first optical element for synthesizing optical paths of observation excitation laser light from the first and second laser light sources;
First scanning means for two-dimensionally scanning on the specimen the excitation laser beam for observation of the optical path synthesized by the first optical element;
A third laser light source for generating stimulation laser light having a multiphoton excitation wavelength;
Second scanning means for two-dimensionally scanning the stimulation laser beam on the specimen;
A second optical element disposed at an intersection position of the optical paths from the first and second scanning means and synthesizing the optical paths of the laser beams from the first and second scanning means , and
The first optical element has a wavelength characteristic that reflects one of the wavelength of the observation laser light from the first laser light source and the wavelength of the observation excitation laser light from the second laser light source and transmits the other. Yes,
The second optical element reflects one of the wavelength of the observation excitation laser light from the first and second laser light sources and the wavelength of the stimulation laser light from the third laser light source and reflects the other. A scanning optical observation apparatus characterized by having a wavelength characteristic to transmit .

When the wavelengths of the respective laser beams from the first, second, and third laser light sources are λ1, λ2, and λ3, and the wavelength of the fluorescence generated from the sample is λf1,
λ1 <λ3 <λ2 and λf1 <λ3,
2. The scanning optical observation apparatus according to claim 1, wherein the second optical element has a characteristic of reflecting one of the wavelengths λ3 and the other wavelengths λ1, λ2, and λf1 and transmitting the other. .

Furthermore, a first fluorescence detecting means having a confocal pinhole is provided on an optical path branched between the first scanning means and the first optical element,
The second optical element is further excited by the observation excitation laser light having the one-photon excitation wavelength of the first laser light source or the multi-photon excitation wavelength of the second laser light source, and the fluorescence wavelength emitted from the specimen. 3. The scanning optical observation apparatus according to claim 1, wherein the scanning optical observation apparatus has a wavelength characteristic of reflecting or transmitting the light toward the first scanning unit .

A second fluorescence detecting means having no confocal pinhole;
Fluorescent wavelength emitted from the specimen by multiphoton excitation by the observation excitation laser light from the second laser light source, which is detachably arranged in the optical path from the first scanning means to the specimen. A third optical element having wavelength characteristics reflected or transmitted toward the detection means;
The scanning optical observation apparatus according to claim 3, comprising:

The scanning optical microscope according to claim 4, wherein the third optical element is disposed on an optical path between the specimen and the second optical element .

The third optical element reflects or transmits the wavelength of each laser beam from the second and third laser light sources toward the specimen, and the wavelength of the fluorescence excited by the multiphoton is the second wavelength. 6. The scanning optical apparatus according to claim 5 , wherein the scanning optical apparatus has wavelength characteristics that are transmitted or reflected toward the fluorescence detection means .

The specimen emits a first fluorescence and a second fluorescence having different wavelengths as the multiphoton excited fluorescence,
The third optical element reflects or transmits the wavelength of the respective laser beams from the second and third laser light sources toward the sample, and one of the first fluorescence and the second fluorescence. 6. The scanning optical system according to claim 5, wherein said scanning optical system has a wavelength characteristic of transmitting or reflecting the light toward the second fluorescence detecting means and reflecting or transmitting the other fluorescence toward the second optical element. apparatus.

5. The scanning optical observation apparatus according to claim 4, wherein the third optical element is disposed in an optical path between the second optical element and the first scanning unit.

The third optical element reflects or transmits the wavelength of the laser light from the second laser light source toward the sample, and directs the wavelength of the multiphoton excited fluorescence toward the second detection unit. 9. The scanning optical observation apparatus according to claim 8, wherein the scanning optical observation apparatus has a wavelength characteristic of transmitting or reflecting.

The scanning optical according to any one of claims 4 to 9, wherein the third optical element is retracted from the optical path when the sample is irradiated with laser light from the first laser light source. Observation device.

2. The scanning optical observation apparatus according to claim 1, wherein a stimulation laser beam having a multiphoton excitation wavelength from the third laser light source can be used as an observation excitation laser beam having a multiphoton excitation wavelength.

The scanning optical observation apparatus according to claim 1, wherein the first to third optical elements are dichroic mirrors.