# US 11,308,688 B2 · Spectrally-resolving high-resolution 3D localization microscopy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,308,688 B2 - Document ID: US11308688B2 - Status: Granted - Kind: B2 - Application number: 16/573,869 - Filed: 2019-09-17 - Published: 2020-03-19 - Granted: 2022-04-19 - Earliest claimed: 2018-09-17 - Assignees: Carl Zeiss Microscopy GmbH - Applicants: Carl Zeiss Microscopy GmbH - Inventors: Thomas Kalkbrenner; Michael Goelles - Web page: https://patentgrep.com/patent/US11308688B2 ## CPC - G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} - G06T 17/00: Three-dimensional [3D] modelling for computer graphics - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G02B 5/04: Prisms - G02B 21/008: {Details of detection or image processing, including general computer control} - G02B 21/06: Means for illuminating specimens - G02B 27/0025: {for optical correction, e.g. distorsion, aberration} - 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)} - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A localization microscope including an imaging device emitting sample light from a focal plane into an image plane, including an optical-manipulation device for depth-dependent influencing of a point-spread function of the imaging and influencing the point-spread function of the imaging such that a point emitter is imaged in the image plane into an image that is rotationally asymmetrically distorted. A form of the distortion depends on the location of the point emitter with respect to the focal plane and a wavelength of the sample light. The optical manipulation device includes first and second anisotropy elements that anisotropically influence the point spread function to produce rotational asymmetry of the point emitter image. The elements are arranged one behind the other in the imaging direction, with anisotropy axes at an angle to one another. Both elements have differing neutral wavelength at which they do not anisotropically influence the point spread. ## Claims ### Claim 1 A localization microscope, comprising: an imaging device for imaging a sample, which emits sample light from a focal plane into an image plane, wherein the imaging device comprises an optical manipulation device that is configured for a depth-dependent influencing of a point spread function of the imaging and influences the point spread function of the imaging such that a point emitter is imaged in the image plane into a point emitter image that is rotationally asymmetrically distorted, wherein at least one of form and orientation of the rotationally asymmetric distortion of the point emitter image depend on a location of the point emitter with respect to the focal plane and a wavelength of the sample light, and wherein the optical manipulation device comprises a first and a second anisotropy element that anisotropically influence in each case the point spread function of the imaging to produce rotational asymmetry of the point emitter image and in each case comprise an anisotropy axis, and wherein: the two anisotropy elements are arranged one behind the other in an imaging direction and their anisotropy axes are located at an angle with respect to one another, both anisotropy elements have a neutral wavelength at which they do not anisotropically influence the point spread function of the imaging, and the neutral wavelengths differ. ### Claim 2 (depends on 1) The localization microscope as claimed in claim 1, wherein the anisotropy elements are formed as a phase element which has a phase-influencing effect at the neutral wavelength of the other anisotropy element and does not have one at the neutral wavelength. ### Claim 3 (depends on 2) The localization microscope as claimed in claim 2, wherein each phase element is embodied as a wedge pair made from two glasses with different dispersion properties, wherein refractive indices and wedge angles are selected such that a wedge angle pair at the neutral wavelength produces direct vision, and otherwise not. ### Claim 4 (depends on 1) The localization microscope as claimed in claim 1, wherein the anisotropy axes are situated at an angle of between 80 and 100 degrees with respect to one another. ### Claim 5 (depends on 4) The localization microscope of claim 4, wherein the anisotropy axes are situated at an angle of 90 degrees with respect to one another. ### Claim 6 (depends on 1) The localization microscope as claimed in claim 1, wherein the anisotropy elements are embodied in each form as an anamorphic lens that has no cylinder effect at the neutral wavelength. ### Claim 7 (depends on 6) The localization microscope as claimed in claim 6, wherein the anisotropy axes are situated at an angle of between 35 and 55 degrees with respect to one another. ### Claim 8 (depends on 7) The localization microscope as claimed in claim 7, wherein the anisotropy axes are situated at an angle of 45 degrees with respect to one another. ### Claim 9 A method for localization microscopy, comprising: emitting sample light from a sample and imaging the sample from a focal plane into an image plane, influencing a point spread function of the imaging in a depth-dependent fashion using an optical manipulation device such that a point emitter is imaged in the image plane into a point emitter image that is rotationally asymmetrically distorted, wherein at least one of form and orientation of the rotationally asymmetric distortion of the point emitter image depend on a location of the point emitter with respect to the focal plane and a wavelength of the sample light, and using a first and a second anisotropy element in the optical manipulation device to anisotropically influence a point spread function of the imaging to produce rotational asymmetry of the point emitter image, each of the first and the second anisotropy elements comprising an anisotropy axis, and wherein: the first and the second anisotropy elements are arranged one behind the other in an imaging direction and with their respective anisotropy axes at an angle with respect to one another, and both the first and the second anisotropy elements have a neutral wavelength at which they do not anisotropically influence the point spread function of the imaging, and the neutral wavelengths differ from one another. ### Claim 10 (depends on 9) The method as claimed in claim 9, further comprising using, for each anisotropy element, a phase element which has a phase-influencing effect at the neutral wavelength of the other anisotropy element and does not have one at the neutral wavelength. ### Claim 11 (depends on 10) The method as claimed in claim 10, further comprising using, for each phase element, a wedge pair made from two glasses with different dispersion properties, including selecting refractive indices and wedge angles such that a wedge angle pair at the neutral wavelength produces direct vision, and otherwise not. ### Claim 12 (depends on 10) The method as claimed in claim 10, wherein the anisotropy axes are situated at an angle of between 80 and 100 degrees with respect to one another. ### Claim 13 (depends on 12) The method as claimed in claim 12, wherein the anisotropy axes are situated at an angle of 90 degrees with respect to one another. ### Claim 14 (depends on 11) The method as claimed in claim 11, further comprising using, for each anisotropy element, an anamorphic lens that has no cylinder effect at the neutral wavelength. ### Claim 15 (depends on 14) The method as claimed in claim 14, wherein the anisotropy axes are situated at an angle of between 35 and 55 degrees with respect to one another. ### Claim 16 (depends on 15) The method as claimed in claim 15, wherein the anisotropy axes are situated at an angle of 45 degrees with respect to one another.