Microscope, sheet-illumination microscope, and microscope-image acquiring method
View Patent ↗A microscope apparatus includes a detection optical system that detects fluorescence produced in a specimen immersed in a medium having a different refractive index from air and accommodated in a container and acquires a fluorescence image. An illumination device focuses excitation light emitted from an excitation-light source, in the form of a plane along a plane intersecting an optical axis of the detection optical system and makes the planar excitation light incident on the specimen in the container. A driving unit moves the specimen in a direction of an optical axis of a cylindrical lens. An illumination control unit adjusts a focal position of the illumination device based on a movement amount of the specimen moved by the driving unit such that an air-equivalent length along an optical path of the excitation light from the illumination device to the optical axis of the detection optical system remains constant.
1. A microscope comprising:
a detection optical system that detects fluorescence produced in a specimen immersed in a medium having a different refractive index from air and accommodated in a container, and that acquires a fluorescence image;
a sheet-illumination optical system that focuses excitation light emitted from a light source in the form of a plane along a plane intersecting an optical axis of the detection optical system and that makes the planar excitation light incident on the specimen in the container;
a driving unit that moves the specimen in a direction of an optical axis of the sheet-illumination optical system;
arithmetic means for calculating a shift amount for a focal position of the sheet-illumination optical system with respect to a position of the optical axis of the detection optical system based on the refractive index of the medium, when the specimen is moved by the driving unit thereby causing a change in an air-equivalent length along an optical path of the excitation light from the sheet-illumination optical system to the optical axis of the detection optical system; and
a focal-position adjusting unit that adjusts the focal position of the sheet-illumination optical system based on a value calculated by the arithmetic means.
2. The microscope according to claim 1 , wherein the driving unit moves the specimen, together with the container, in the direction of the optical axis of the sheet-illumination optical system.
3. The microscope according to claim 1 , wherein:
the container is fixed to a microscope body in which the detection optical system is provided; and
the driving unit moves the specimen in the container, which is fixed to the microscope body, in the direction of the optical axis of the sheet-illumination optical system.
4. The microscope according to claim 1 , wherein the focal-position adjusting unit adjusts the focal position of the sheet-illumination optical system based on the movement amount of the specimen moved by the driving unit such that the air-equivalent length is maintained to be constant.
5. The microscope according to claim 1 , wherein the focal-position adjusting unit adjusts the focal position of the sheet-illumination optical system by moving the sheet-illumination optical system in the direction of the optical axis thereof.
6. The microscope according to claim 1 , wherein the driving unit is capable of moving the specimen in two-dimensional directions along an incident plane of the excitation light.
7. The microscope according to claim 1 , further comprising an image processing unit that generates a composite image by arraying, at respective coordinates of the specimen moved to by the driving unit, fluorescence images of the specimen acquired by the detection optical system.
8. A sheet-illumination microscope in which a specimen immersed in a medium having a different refractive index from air is irradiated with illumination light in the form of a sheet from a direction intersecting an observation optical axis and is observed, the sheet-illumination microscope comprising:
an objective lens that collects light produced in the specimen, which is immersed in the medium and is accommodated in a container;
a detector that detects the light collected by the objective lens;
a sheet-illumination optical system that focuses, in the form of a plane, illumination light emitted from a light source and that makes the planar illumination light incident on the specimen in the container along a plane intersecting an optical axis of the objective lens;
a focal-point adjusting mechanism that moves a focal position of the sheet-illumination optical system in a direction of an optical axis of the sheet-illumination optical system;
a stage that can move at least one of the container and the specimen in the direction of the optical axis of the sheet-illumination optical system; and
a controller that (i) calculates a shift amount for the focal position of the sheet-illumination optical system with respect to a position of the optical axis of the objective lens based on the refractive index of the medium, when the stage is moved thereby causing a change in an air-equivalent length along an optical path of excitation light from the sheet-illumination optical system to the optical axis of the objective lens, and (ii) controls the focal-point adjusting mechanism to adjust the focal position of the sheet-illumination optical system based on the shift amount having been calculated.
9. The sheet-illumination microscope according to claim 8 , wherein the stage has the container mounted thereon and moves the specimen together with the container.
10. The sheet-illumination microscope according to claim 8 , wherein the controller performs control so as to adjust the focal position of the sheet-illumination optical system based on a movement amount of the stage such that the air-equivalent length is maintained to be constant.
11. A microscope-image acquiring method comprising:
moving a specimen immersed in a medium having a different refractive index from air and accommodated in a container in a direction along an optical axis of a sheet-illumination optical system, the optical axis of the sheet-illumination optical system intersecting an optical axis of a detection optical system;
focusing, with the sheet-illumination optical system, excitation light emitted from a light source in the form of a plane along a plane intersecting the optical axis of the detection optical system and making the planar excitation light incident on the specimen;
calculating a shift amount for a focal position of the sheet-illumination optical system with respect to a position of the optical axis of the detection optical system based on the refractive index of the medium, when the specimen is moved in the moving thereby causing a change in an air-equivalent length along an optical path of the excitation light from the sheet-illumination optical system to the optical axis of the detection optical system;
adjusting the focal position of the sheet-illumination optical system based on a value calculated in the calculating so as not to be shifted from the position of the optical axis of the detection optical system; and
detecting, with the detection optical system, fluorescence produced in the specimen on which the excitation light is made incident.
12. The microscope-image acquiring method according to claim 11 , wherein the specimen is moved together with the container in the direction along the optical axis of the sheet-illumination optical system.
13. The microscope-image acquiring method according to claim 11 , wherein:
the container is fixed to a microscope body in which the detection optical system is provided; and
the specimen in the container, which is fixed to the microscope body, is moved in the direction along the optical axis of the sheet-illumination optical system.
14. The microscope-image acquiring method according to claim 11 , wherein, in the adjusting the focal position, the focal position of the sheet-illumination optical system is adjusted based on a movement amount of the specimen such that the air-equivalent length is maintained to be constant.