# US 11,307,397 B2 · High-resolution confocal microscope > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,307,397 B2 - Document ID: US11307397B2 - Status: Granted - Kind: B2 - Application number: 16/650,252 - Filed: 2018-09-25 - Published: 2021-12-02 - Granted: 2022-04-19 - Earliest claimed: 2017-10-02 - Assignees: Carl Zeiss Microscopy GmbH - Applicants: Carl Zeiss Microscopy GmbH - Inventors: Wolfgang Bathe; Stanislav Kalinin - Web page: https://patentgrep.com/patent/US11307397B2 ## CPC - G02B 21/0076: {arrangements using fluorescence or luminescence} - G02B 21/008: {Details of detection or image processing, including general computer control} - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G02B 21/0032: {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)} - G02B 21/0036: {Scanning details, e.g. scanning stages} - G02B 21/0072: {details concerning resolution or correction, including general design of CSOM objectives} - G02B 27/58: Optics for apodization or superresolution; Optical synthetic aperture systems - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - 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 method for the high-resolution scanning microscopy of a specimen where the specimen is illuminated with illuminating radiation such that the illuminating radiation is focused to a diffraction-limited illuminating spot at a point in or on the specimen. The point is projected in a diffraction-limited manner in a diffraction image onto a flat panel detector having pixels. The flat panel detector, owing to the pixels thereof, have a spatial resolution which resolves a diffraction structure of the diffraction image. The point is shifted relative to the specimen into different scanning positions by an increment which is smaller than the diameter of the illuminating spot and a 3D image is generated. The pixels of the flat panel detector are divided into groups. A pre-calculated raw image is calculated for each group and are unfolded three-dimensionally to generate the image of the specimen. ## Claims ### Claim 1 A method for high-resolution scanning microscopy of a sample, comprising: illuminating the sample with illumination radiation by focusing the illumination radiation to a point in or on the sample to form a diffraction-limited illumination spot comprising a diameter; imaging the point in diffraction-limited manner to a diffraction image comprising a diffraction structure onto a 2D detector, the detector having pixels and a spatial resolution that resolves the diffraction structure of the diffraction image, wherein imaging in diffraction-limited manner comprises a resolution limit; displacing the point relative to the sample into different scanning positions with a scanning increment less than the diameter of the illumination spot; reading data from the 2D detector and assigned an actual scanning position to the data for each of the scanning positions, and generating an image of the sample from the data and from the scanning positions assigned, wherein the generating step comprises: assigning each of the pixels of the 2D detector either to a central group located on an optical axis or to at least one further group that surrounds the central group in ring-shaped fashion, calculating a pre-processed raw image for each group, and combining and deconvoluting the pre-processed raw images to generate the image of the sample having a resolution that is increased beyond the resolution limit. ### Claim 2 (depends on 1) The method as claimed in claim 1, wherein the pre-processed raw images are deconvoluted in three dimensions and a 3D image of the sample is generated. ### Claim 3 (depends on 1) The method as claimed in claim 1, wherein the groups include at least one additional ring-shaped surrounding group, wherein each ring-shaped group has an individual distance from the central group. ### Claim 4 (depends on 1) The method as claimed in claim 1, wherein four or five groups are provided. ### Claim 5 (depends on 1) The method as claimed in claim 1, wherein each pixel is assigned to exactly one of the groups. ### Claim 6 (depends on 1) The method as claimed in claim 1, wherein the pre-processed raw images are calculated by virtue of a two-dimensional deconvolution being carried out for each group. ### Claim 7 (depends on 1) The method as claimed in claim 1, wherein relative displacements of raw image signals contained in the data are calculated, the raw image signals are compensated with respect to displacement and the compensated raw image signals belonging to the same group are added to calculate the pre-processed raw image for this group. ### Claim 8 A microscope for high-resolution scanning microscopy of a sample, comprising: an illumination beam path for illuminating the sample with illumination radiation by focusing the illumination radiation to a point in or on the sample to form a diffraction-limited illumination spot having a diameter; an imaging beam path for diffraction-limited imaging of the point into a diffraction image image comprising a diffraction structure onto an 2D detector, the detector having pixels and a spatial resolution resolving the diffraction structure, wherein diffraction-limited imaging manner comprises a resolution limit; a scanning device for displacing the point relative to the sample into different scanning positions with a scanning increment less than the diameter of the illumination spot, and an evaluation device configured to read data from the 2D detector and to assign an actual scanning position to the data for each scanning position, wherein the evaluation device is further configured to generate an image of the sample from the data and from the scanning positions assigned by assigning each of the pixels of the 2D detector either to a central group located on an optical axis or to at least one further group that surrounds the central group in ring-shaped fashion, calculating a pre-processed raw image for each group, and combining and deconvoluting the pre-processed raw images to generate the image of the sample having a resolution that is increased beyond the resolution limit. ### Claim 9 (depends on 8) The microscope as claimed in claim 8, wherein the evaluation device is configured to deconvolute the pre-processed raw images in three-dimensions in order to generate the image the sample. ### Claim 10 (depends on 8) The microscope as claimed in claim 8, wherein the groups include at least one additional ring-shaped surrounding group, wherein each ring-shaped group has an individual distance from the central group. ### Claim 11 (depends on 8) The microscope as claimed in claim 8, wherein four or five groups are provided. ### Claim 12 (depends on 8) The microscope as claimed in claim 8, wherein each pixel is assigned to exactly one of the groups. ### Claim 13 (depends on 8) The microscope as claimed in claim 8, wherein the evaluation device is further configured to calculate the pre-processed raw images by carrying out a two-dimensional deconvolution for the groups.