Specimen observation apparatus

• US 10,642,013 B2
• Filed: 04/23/2015
• Issued: 05/05/2020
• Est. Priority Date: 04/30/2014
• Status: Active Grant

• Current Assignee: Evident Corporation
• Original Assignee: Olympus Corporation
• Inventors: Kitagawa, Hisao
• Primary Examiner(s): Uhl, Lindsay J
• Application Number:
  US14/694,555
• Publication Number:
  US 20150316757A1
• Time in Patent Office: 1,839 Days
• Field of Search: None
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0044 moving apertures, e.g. Nipkow disks, rotating lens arrays
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G02B 27/58 Optics for apodization or superresolution; Optical synthetic aperture systems

Abstract

A superior superresolution image can be generated. Provided is a specimen observation apparatus including an objective lens that radiates excitation laser light emitted from a light source onto a specimen; and a main controller that obtains a plurality of sets of image data of the same region on the specimen by repeatedly detecting fluorescence from the same region on the specimen irradiated with the excitation laser light by the objective lens, and that emphasizes high-frequency components in an addition image data obtained by adding the obtained plurality of sets of image data of the same region on the specimen.

Claims

1. A specimen observation apparatus comprising:

an objective lens that radiates laser light emitted from a light source onto a specimen;

an image-data obtaining portion that is configured to detect return light from the specimen irradiated with the laser light by the objective lens and that obtains an image; and

a computer that is configured to;

repeatedly detect the return light from a same region on the specimen with the image-data obtaining portion and obtain a plurality of images for the same region from the repeated detection;

add the plurality of images for the same region, obtained by the image-data obtaining portion, so as to obtain added image data; and

subject the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data to generate a superresolution image.

2. The specimen observation apparatus according to claim 1, further comprising:

a confocal aperture portion that has an opening at a position that is optically conjugate with a focal position of the objective lens; and

a confocal lens that focuses return light from the specimen irradiated with the laser light by the objective lens and that projects a spot of the return light at the opening position of the confocal aperture portion,

wherein the opening of the confocal aperture portion has a diameter smaller than the diameter of the spot of return light projected by the confocal lens.

3. The specimen observation apparatus according to claim 1, further comprising:

a scanner that scans the laser light, irradiated onto the specimen by the objective lens, on the specimen in a main scanning direction and a sub-scanning direction that intersects therewith,

wherein the computer repeats an operation in which, after the laser light is repeatedly scanned on a same scanning line in the main scanning direction by the scanner for a prescribed number of times, a position thereof in the sub-scanning direction is changed so as to move to a next scanning line, and a plurality of images for the same scanning line are obtained by the image-data obtaining portion.

4. The specimen observation apparatus according to claim 1, further comprising:

the light source that emits line-shaped laser light,

wherein the image-data obtaining portion includes an image-acquisition device formed in the shape of a line, which acquires an image of the return light that returns from the specimen irradiated with the line-shaped laser light.

5. The specimen observation apparatus according to claim 1, further comprising:

a mask that has openings in the shape of slits or pinholes arranged periodically and that restricts the laser light incident on the objective lens; and

a motor that rotates the mask about a center axis thereof,

wherein the image-data obtaining portion includes a two-dimensional image-acquisition device that acquires an image of the return light returning from the specimen.

9. The specimen observation apparatus according to claim 1, wherein the image-data obtaining portion includes a multialkali photomultiplier tube.

10. The specimen observation apparatus according to claim 1, wherein the image-data obtaining portion includes a gallium arsenide phosphide photomultiplier tube.

11. The specimen observation apparatus according to claim 1, wherein the image-data obtaining portion includes an image sensor.

12. The specimen observation apparatus according to claim 1, wherein the image-data obtaining portion includes the computer.

13. The specimen observation apparatus according to claim 4, wherein the image-acquisition device includes a CCD line camera or an optical multichannel analyzer.

14. The specimen observation apparatus according to claim 5, wherein the two-dimensional image-acquisition device includes a CCD or a CMOS device.

15. The specimen observation apparatus according to claim 2, wherein the confocal aperture portion comprises an aperture.

16. The specimen observation apparatus according to claim 2, wherein the confocal aperture portion comprises a plurality of pin holes having different diameters and which are switchable by the computer to be selectively disposed at the position that is optically conjugate with the focal position of the objective lens.

22. The specimen observation apparatus according to claim 1, wherein:

said same region on the specimen is one of a plurality of regions on the specimen which together form an entire region of the specimen to be imaged; and

the computer is configured to;

repeatedly detect return light from each of said plurality of regions, respectively, and obtain, for each same region among the plurality of regions, a plurality of images from the repeated detection;

respectively add the plurality of images for said each same region, obtained by the image-data obtaining portion, so as to obtain added image data for said each same region; and

subject the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data for said each same region to generate the superresolution image.

6. A specimen observation apparatus comprising:

a scanning mirror that scans laser light emitted from a light source on a specimen;

an objective lens that radiates the laser light scanned by the scanning mirror onto the specimen;

a light detector that detects return light from the specimen irradiated with the laser light and that outputs a signal according to a light level thereof; and

a controller that generates an image of the specimen by using the signal output from the light detector,

wherein the controller performs processing for generating a plurality of images for a same position by controlling the scanning mirror so as to repeatedly scan the laser light a plurality of times at the same position on the specimen, processing for adding the generated plurality of images for the same position to obtain added image data, and processing for subjecting the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data to generate a superresolution image.

7. The specimen observation apparatus according to claim 6,

wherein the scanning mirror includes at least one galvanometer mirror that deflects the laser light in a main scanning direction and a sub-scanning direction, which intersect each other,

wherein the controller controls the scanning mirror so as to repeatedly perform an operation in which, after the laser light is repeatedly scanned on a same scanning line in the main scanning direction a prescribed number of times, a position thereof in the sub-scanning direction is changed so as to move to a next scanning line.

8. A specimen observation apparatus comprising:

an objective lens that radiates excitation light emitted from a light source onto a specimen;

a mask that is disposed on a light-source side of the objective lens and that has slit-shaped openings or pinholes which are periodically arranged;

a motor that rotates the mask about a prescribed rotation axis;

a two-dimensional image-acquisition device that acquires an image of return light from the specimen irradiated with the excitation light via the rotating mask; and

a controller that performs processing for repeatedly obtaining and adding the images of a same region on the specimen, obtained by the two-dimensional image-acquisition device, so as to obtain added image data, and processing for subjecting the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data to generate a superresolution image.

17. A specimen observation apparatus comprising:

an objective lens that radiates laser light emitted from a light source onto a specimen;

a scanner that scans the laser light, irradiated onto the specimen by the objective lens, on the specimen;

an image-data obtaining portion that is configured to detect return light from the specimen irradiated with the laser light by the objective lens and that obtains an image; and

a computer that is configured to;

repeatedly detect the return light from a same region on the specimen with the image-data obtaining portion and obtain a plurality of images for the same region from the repeated detection;

add the plurality of images for the same region, obtained by the image-data obtaining portion, so as to obtain added image data; and

subject the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data to generate a superresolution image,

wherein;

the image-data obtaining portion comprises a light detector that detects return light from the specimen and that outputs a light intensity signal according to a light level thereof,

the image-data obtaining portion generates the image data of the specimen by converting the light intensity signal output from the light detector to luminance information for each pixel, according to a scanning position of the scanner, and

the computer performs processing for generating the plurality of images for the same region by repeatedly scanning the laser light a plurality of times at the same region on the specimen.

18. The specimen observation apparatus according to claim 17, further comprising:

a confocal aperture portion that has an opening at a position that is optically conjugate with a focal position of the objective lens; and

a confocal lens that focuses return light from the specimen irradiated with the laser light by the objective lens and that projects a spot of the return light at the opening position of the confocal aperture portion,

wherein the opening of the confocal aperture portion has a diameter smaller than the diameter of the spot of return light projected by the confocal lens.

19. The specimen observation apparatus according to claim 17,

wherein the scanner scans the laser light on the specimen in a main scanning direction and a sub-scanning direction that intersects therewith,

wherein the computer repeats an operation in which, after the laser light is repeatedly scanned on a same scanning line in the main scanning direction by the scanner for a prescribed number of times, a position thereof in the sub-scanning direction is changed so as to move to a next scanning line, and a plurality of images for the same scanning line are obtained by the image-data obtaining portion.

20. A specimen observation apparatus comprising:

a scanning mirror that scans laser light emitted from a light source on a specimen;

an objective lens that radiates the laser light scanned by the scanning mirror onto the specimen;

a light detector that detects return light from the specimen irradiated with the laser light and that outputs a light intensity signal according to a light level thereof; and

a controller that generates an image of the specimen by using the light intensity signal output from the light detector,

wherein the controller performs processing for generating a plurality of images for a same position by controlling the scanning mirror so as to repeatedly scan the laser light a plurality of times at the same position on the specimen to convert the light intensity signal output from the light detector to luminance information for each pixel, according to a scanning position of the scanning mirror, processing for adding the generated plurality of images for the same position to obtain added image data, and processing for subjecting the added image data, serving as a raw image, to further processing to emphasize high-frequency components in the obtained added image data to generate a superresolution image.

21. The specimen observation apparatus according to claim 20, wherein the scanning mirror includes at least one galvanometer mirror that deflects the laser light in a main scanning direction and a sub-scanning direction, which intersect each other,

wherein the controller controls the scanning mirror so as to repeatedly perform an operation in which, after the laser light is repeatedly scanned on a same scanning line in the main scanning direction a prescribed number of times, a position thereof in the sub-scanning direction is changed so as to move to a next scanning line.