INCREASED DEPTH-RESOLUTION MICROSCOPY

• US 20130302905A1
• Filed: 11/11/2011
• Published: 11/14/2013
• Est. Priority Date: 11/16/2010
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: Kalkbrenner, Thomas, Lippert, Helmut, Kleppe, Ingo
• Granted Patent: US 9,201,011 B2
• Time in Patent Office: Days
• US Class Current: 436/172
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6428 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 Class Codes: G01N 21/6447 by visual observation
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• 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 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.

Claims

1. A method for high-resolution luminescence microscopy of a sample marked with marking molecules that can be activated and subsequently excited to emit particular luminescent radiation, wherein the method comprises:

a) repeated activation and excitation of a subset of the marking molecules to emit luminescent radiation, at least some of the luminescent marking molecules being at least at a distance from the luminescent marking molecules immediately adjacent thereto greater than or equal to a length which results from a predetermined optical resolution;

b) repeated imaging of the sample along a depth direction and with the predetermined optical resolution;

c) production of individual images from the imaging operations from step b), wherein geometrical locations of the luminescent marking molecules in the individual images are determined with a spatial resolution which is increased above the predetermined optical resolution; and

wherein

d) the activation and/or excitation in step a) is effected with radiation which is introduced 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, when seen in the depth direction, arranged in such a manner that said regions are behind one another and overlap only partially, and wherein

e) separate images of the sample are recorded in step b) for activation and/or excitation of each of the at least two regions in order to obtain a depth information relating to the luminescent marking molecules from the separate images.

2. The method according to claim 1, wherein in step e), those luminescent marking molecules that appear in both of the at least two separate images are filtered out.

3. The method according to claim 2, wherein luminescence intensities of the filtered-out marking molecules in the at least two images are compared and a depth specification is derived therefrom.

4. The method according to claim 1, wherein, in step d), the radiation is introduced in the form of at least one light sheet introduced transversely in relation to the depth direction and the thickness of which defines the extent, and wherein the thickness of the light sheet is diffraction-limited.

5. The method according to claim 4, wherein the light sheet (30, 31) is displaced transversely in relation to the depth direction, relative to the sample into at least two positions.

6. The method according to claim 4, wherein two spaced-apart light sheets are used, and wherein the light sheets are switched on and off alternately or are differentially modulated.

7. The method according to claim 1, wherein, in step d), the radiation along the depth direction is introduced in the form of at least one pair of diffraction-limited foci, wherein a first focus of the pair is focussed into a first of two planes, and a second focus of the pair is focussed into a second of the two planes, the foci displaced along the respective planes in order to form the two regions, and wherein the depth of field of the focussing defines the extent of the regions.

8. 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, they 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 length which results from a predetermined optical resolution;

b) an imaging device for repeated imaging of the sample along a depth direction and with the predetermined optical resolution;

c) a control and evaluation device which produces individual images from the imaging operations 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 behind one another and overlap only partially; and

wherein

e) the imaging device records 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.

9. The microscope according to claim 8, 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.

10. The microscope according to claim 9, wherein the illumination beam path includes a device that displaces the light sheet, transversely in relation to the depth direction, into at least two positions.

11. The microscope according to claim 9, wherein the illumination beam path has two spaced-apart light sheets, the light sheets being switched on and off alternately or differentially modulated.

12. The microscope according to claim 9, wherein the illumination beam path introduces the activation and/or excitation radiation along the depth direction in the form of at least one pair of diffraction-limited foci, focuses a first focus of the pair of diffraction-limited foci into a first of two planes and focuses a second focus (42) of the pair of diffraction-limited foci into a second of the two planes, and displaces the foci along the respective planes in order to form the two regions, wherein the depth of field of the focussing defines the extent of the regions.

13. The microscope according to claim 12, wherein the illumination beam path comprises a mini-lens array arranged on a rotating disc.

14. (canceled)