Increased depth-resolution microscopy

Patent metadata

Field	Value
Publication number	US-9201011-B2
Application number	US-201113885979-A
Country	US
Kind code	B2
Filing date	Nov 11, 2011
Priority date	Nov 16, 2010
Publication date	Dec 1, 2015
Grant date	Dec 1, 2015

Abstract

A method for high-resolution luminescence microscopy of a sample marked with marking molecules that can be activated to excite particular luminescent radiation, including: repeated activation of a subset of the marking molecules to emit luminescent radiation; repeated imaging of the sample along a depth direction and with a predetermined optical resolution; and producing images from the repeated imaging. Locations of the marking molecules are determined with a spatial resolution that is increased above the predetermined optical resolution. Activation of the marking molecules can be through radiation introduced into multiple regions, each extending along a plane substantially perpendicular to the depth direction. The regions can be arranged so that the regions are behind one another and overlap only partially. Separate images of the sample may be recorded for activation in each of the regions in order to obtain depth information relating to the marking molecules from the separate images.

First claim

The invention claimed is:

1. A microscope for high-resolution luminescence microscopy of a sample marked with marking molecules that can be activated in such a manner that, once activated, the marking molecules can be excited to emit particular luminescent radiation, wherein the microscope comprises:
a) an illumination beam path formed for repeated activation and excitation of a subset of the marking molecules to emit luminescent radiation at least some of the luminescent marking molecules at least at a distance from the luminescent marking molecules immediately adjacent thereto greater than or equal to a minimum resolvable length which results from a predetermined optical resolution;
b) an imaging device including an objective and a detector, the imaging device being configured to repeatedly image the sample along a depth direction and with the predetermined optical resolution;
c) a control and evaluation device, including a computer, operably coupled to the imaging device and programmed with an algorithm that produces individual images based on data received from the imaging device and determines the geometrical locations of the luminescent marking molecules in the individual images with a spatial resolution which is increased above the predetermined optical resolution; wherein
d) the illumination beam path introduces the activation and/or excitation radiation into at least two regions, the regions each extending along a plane substantially perpendicular to the depth direction, the regions having a predetermined extent in the depth direction, and the regions arranged in such a manner that said regions are one behind another in the depth direction and overlap only partially; and wherein
e) the imaging device is configured to capture separate images of the sample for activation and/or excitation of each of the at least two regions and the control and evaluation device obtains a depth information relating to the marking molecules from the separate images.

2. The microscope according to claim 1 , wherein the illumination beam path introduces the activation and/or excitation radiation in the form of at least one light sheet that lies transversely in relation to the depth direction and the thickness of which defines the extent, the thickness of the light sheet being diffraction-limited.

3. The microscope according to claim 2 , wherein the illumination beam path includes an optical element that displaces the light sheet, transversely in relation to the depth direction, into at least two positions.

4. The microscope according to claim 2 , wherein the illumination beam path has two spaced-apart light sheets, the light sheets being switched on and off alternately or differentially modulated.

5. The microscope according to claim 2 , wherein the illumination beam path focuses the activation and/or excitation radiation along the depth direction into two light sheets that each lie transversely in relation to the depth direction in two respective planes, each of the two respective planes being oriented transversely to the depth direction, focuses a first focus of a pair of diffraction-limited foci into a first of the two planes and focuses a second focus of the pair of diffraction-limited foci into a second of the two planes, and displaces the foci along the two respective planes in order to form the two regions, wherein the depth of field of the focussing defines the extent of the regions.

6. The microscope according to claim 5 , wherein the illumination beam path comprises a mini-lens array arranged on a rotating disc.

7. The microscope according to claim 3 , wherein the optical element that displaces the light sheet comprises a scanning mirror.

8. The microscope according to claim 3 , wherein the optical element that displaces the light sheet comprises a Nipkov disc.

9. The microscope according to claim 3 , wherein the optical element that displaces the light sheet comprises a diffractive optical element.

10. The microscope according to claim 3 , wherein the optical element that displaces the light sheet comprises a combination of prisms.

Assignees

• Kalkbrenner Thomas
• Lippert Helmut
• Kleppe Ingo
• Zeiss Carl Microscopy Gmbh

Inventors

• Kalkbrenner Thomas
• Lippert Helmut
• Kleppe Ingo

Classifications

• G01N21/6447 Primary

  by visual observation · CPC title

• G01N21/64

  Fluorescence; Phosphorescence · CPC title

• G02B21/367

  providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title