Laser scanning microscope apparatus

Patent metadata

Field	Value
Publication number	US-2016306152-A1
Application number	US-201615132053-A
Country	US
Kind code	A1
Filing date	Apr 18, 2016
Priority date	Apr 20, 2015
Publication date	Oct 20, 2016
Grant date	—

Abstract

A laser scanning microscope apparatus includes an irradiation unit including an objective lens, a photodetector unit, an XY-scanning unit, and a Z-scanning unit. The irradiation unit focuses a laser beam with the objective lens to a specimen. The photodetector unit detects light generated from a position irradiated with the laser beam focused. The XY-scanning unit scans the laser beam in an X-direction perpendicular to an optical axis of the objective lens and in a Y-direction perpendicular to the optical axis and the X-direction. The Z-scanning unit scans the laser beam in a Z-direction parallel to the optical axis. When acquiring XY-two-dimensional image data by detecting the light while scanning the irradiated position in the X-direction and the Y-direction, the apparatus detects the light while scanning the irradiated position also in the Z-direction.

First claim

What is claimed is:

1 . A laser scanning microscope apparatus comprising:
an irradiation unit, comprising an objective lens, and configured to focus a laser beam with the objective lens to a specimen; a photodetector unit configured to detect light generated from a position irradiated with the focused laser beam; an XY-scanning unit configured to scan the laser beam applied to the specimen with the irradiation unit in an X-direction perpendicular to an optical axis of the objective lens and in a Y-direction perpendicular to the optical axis and the X-direction; and a Z-scanning unit configured to scan the laser beam applied to the specimen with the irradiation unit in a Z-direction parallel to the optical axis of the objective lens, wherein, when acquiring XY-two-dimensional image data in which information of the detected light is stored for each pixel corresponding to an X-coordinate and a Y-coordinate of the irradiated position by detecting the light while scanning the irradiated position in the X-direction and the Y-direction with the XY-scanning unit, the apparatus is configured to detect the light while scanning the irradiated position also in the Z-direction with the Z-scanning unit to acquire XY-two-dimensional image data including at least two pixels in which information of the light generated from irradiated positions at different Z-coordinates is individually stored.

2 . The laser scanning microscope apparatus according to claim 1 , wherein the apparatus is configured to detect the light while scanning the irradiated position in the X-direction and the Y-direction with the XY-scanning unit and while scanning the irradiated position also in the Z-direction with the Z-scanning unit.

3 . The laser scanning microscope apparatus according to claim 1 , further comprising a wavelength changing unit configured to change a wavelength of the laser beam;
wherein the apparatus is configured to acquire XY-two-dimensional image data including at least two pixels in which information of the light generated due to the different wavelengths of the laser beam is individually stored by detecting the light while changing the wavelength of the laser beam with the wavelength changing unit while the irradiated position is scanned in at least one of the X-direction, the Y-direction, and the Z-direction.

4 . The laser scanning microscope apparatus according to claim 3 , wherein the wavelength changing unit is configured to periodically change the wavelength of the laser beam.

5 . The laser scanning microscope apparatus according to claim 1 ,
wherein the XY-scanning unit is configured to periodically scan the irradiated position in the X-direction and the Y-direction, and wherein the Z-scanning unit is configured to periodically scan the irradiated position in the Z-direction.

6 . The laser scanning microscope apparatus according to claim 5 , wherein the XY-scanning unit is configured to scan the irradiated position in the X-direction and the Y-direction at longer intervals than intervals at which the Z-scanning unit is configured to scan the irradiated position in the Z-direction.

7 . The laser scanning microscope apparatus according to claim 3 ,
wherein the irradiation unit comprises:
a first light source configured to emit a first laser beam;
a second light source configured to emit a second laser beam; and
a multiplexer configured to multiplex the first laser beam and the second laser beam to form the laser beam,
wherein the wavelength changing unit is configured to change a difference in wavelength between the first laser beam and the second laser beam.

8 . The laser scanning microscope apparatus according to claim 1 , wherein the light comprises light including stimulated Raman scattered light or coherent anti-Stokes Raman scattered light.

9 . The laser scanning microscope apparatus according to claim 1 , wherein the Z-scanning unit is configured to change the Z-coordinate of a starting point of the scanning of the irradiated position in the Z-direction based on an intensity of the light detected by the scanning of the irradiated position in the Z-direction.

10 . The laser scanning microscope apparatus according to claim 5 , wherein the Z-scanning unit is configured to periodically scan the irradiated position in the Z-direction by vibrating the specimen in the Z-direction.

11 . The laser scanning microscope apparatus according to claim 3 , wherein the XY-two-dimensional image data comprises spectral image data in which spectral data storing an intensity of the light with respect to the wavelength of the laser beam is stored for each of the pixels.

12 . The laser scanning microscope apparatus according to claim 11 , further comprising a data processing unit configured to process the spectral image data by a multivariate analysis.

13 . The laser scanning microscope apparatus according to claim 1 , wherein the light that the photodetector unit detects comprises at least one selected from transmitted light, reflected light, and scattered light generated from the irradiated position.

Assignees

• Canon Kk

Inventors

• Makara Koji
• Otsuka Yoichi
• Kyogaku Masafumi
• Hashimoto Hiroyuki

Classifications

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/65

  Raman scattering · CPC title

• G02B21/0032

  Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers (G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06) · CPC title

• G02B21/0036 Primary

  Scanning details, e.g. scanning stages · CPC title

• G02B21/008 Primary

  Details of detection or image processing, including general computer control · CPC title