LIGHT SCANNING MICROSCOPE AND MICROSCOPY METHOD

• US 20130314758A1
• Filed: 05/24/2013
• Published: 11/28/2013
• Est. Priority Date: 05/25/2012
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: Hellmich, Wibke, Krampert, Gerhard, Netz, Ralf, Langhorst, Matthias
• Granted Patent: US 9,304,307 B2
• Time in Patent Office: Days
• US Class Current: 359/234
• CPC Class Codes: G02B 21/0044 moving apertures, e.g. Nipkow disks, rotating lens arrays
• CPC Class Codes: G02B 21/0052 Optical details of the image generation
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control

Abstract

A light scanning microscope with an illumination module generates several illumination beams and moves them, in each case as a spot, in a predefined region of a sample to excite sample radiation. A detector module for confocal detection of the sample radiation excited by each spot includes a first detector, an imaging lens system, having an optical axis, for imaging the predefined region along an imaging beam path running from the sample as far as the first detector, and a rotatable diaphragm with several pinholes located in a pinhole plane. The diaphragm, upon rotation, may be located at least partially in the imaging beam path for confocal detection. A second detector may be arranged outside of the imaging beam path. A first beam splitter may be arranged in the imaging beam path between the sample and the diaphragm. The beam splitter deflects sample radiation onto the second detector.

Claims

1. Light scanning microscope, comprising:

an illumination module, which generates several illumination beams and moves them, in each case as a spot, in a predefined region of a sample for the purpose of exciting sample radiation;

a detector module for confocal detection of the sample radiation excited by each spot, the detector module comprising a first detector, an imaging lens system, having an optical axis, for imaging the predefined region along an imaging beam path running from the sample as far as the first detector, and a rotatable diaphragm with several pinholes which are located in a pinhole plane, wherein the diaphragm, upon rotation, is located at least partially in the imaging beam path for the purpose of confocal detection;

a second detector disposed outside of the imaging beam path; and

a first beam splitter disposed in the imaging beam path between the sample and the diaphragm, wherein the beam splitter deflects sample radiation, reflected by the diaphragm, onto the second detector.

2. The light scanning microscope according to claim 1, wherein a second beam splitter, which effects beam-path folding of the imaging beam path, is arranged between the diaphragm and the first detector.

3. The light scanning microscope according to claim 2, wherein the diaphragm comprises a constituent part of the illumination module, and the rotation of the diaphragm effects the movement of the spots, wherein the propagation direction of the radiation for generating the illumination beams is preferably perpendicular to the pinhole plane.

4. The light scanning microscope according to claim 3, wherein the illumination module comprises a disk with several imaging elements, the disk is connected to the diaphragm in a rotationally fixed manner, and each imaging element is associated with exactly one pinhole, such that in each case it focuses the illumination beam into the respective associated pinhole.

5. The light scanning microscope according to claim 1, wherein the diaphragm comprises a constituent part of the illumination module, and the rotation of the diaphragm effects the movement of the spots, wherein the propagation direction of the radiation for generating the illumination beams is generally perpendicular to the pinhole plane.

6. The light scanning microscope according to claim 5, wherein the illumination module comprises a disk with several imaging elements, the disk is connected to the diaphragm in a rotationally fixed manner, and each imaging element is assigned to exactly one pinhole, such that in each case it focuses the illumination beam into the assigned pinhole.

7. The light scanning microscope according to claim 1, wherein the part of the diaphragm which, upon rotation, is arranged at least partially in the imaging beam path is positioned in an intermediate image plane of the imaging lens system.

8. The light scanning microscope according to claim 1, further comprising a detection lens system, which images onto the second detector the part of the diaphragm that is at least partially located in the imaging beam path.

9. The light scanning microscope according to claim 1, wherein the diaphragm comprises a Nipkow disk.

10. The light scanning microscope according to claim 1, wherein the side of the diaphragm that faces towards the first beam splitter is reflective of the sample radiation.

11. The light scanning microscope according to claim 1, further comprising a control unit, to which the signals of the two detectors are supplied, and which takes account of the signals in the generation of a sample image.

12. The light scanning microscope according to claim 11, wherein, for the purpose of determining an optical section of the sample, the control unit is configured to perform a scaled subtraction of the signals of the first and second detectors.

13. The light scanning microscope according to claim 12, wherein, for the purpose of determining a widefield image, the control unit is configured to perform a scaled addition of the signals of the first and second detectors.

14. The light scanning microscope according to claim 11, wherein, for the purpose of determining a widefield image, the control unit is configured to perform a scaled addition of the signals of the first and second detectors.

15. The light scanning microscope according to claim 1, wherein the microscope is configued as a laser scanning microscope.

16. A microscopy method, comprising:

generating several illumination beams;

moving each illumination beam, as a spot, in a predefined region of a sample, to exciting sample radiation;

detectiong the sample radiation excited by each spot confocally by a detector module, the detector module comprising a first detector, an imaging lens system for imaging the predefined region along an imaging beam path running from the sample as far as the detector, and a rotatable diaphragm with several pinholes;

rotationg the diaphragm such that it is located at least partially in the imaging beam path for the purpose of confocal detection,

disposing a second detector outside of the imaging beam path

disposing a beam splitter in the imaging beam path between the sample and the diaphragm; and

deflecting with the beam splitter the sample radiation, coming from the diaphragm, onto the second detector.