Carl Zeiss Microscopy Gmbh

Jörg Ritter

Thomas Kalkbrenner

Helmut Lippert

Jörg Siebenmorgen

Tobias Kaufhold

A three-dimensional high-resolution localization microscopy method including illuminating a sample by excitation radiation to excite fluorescence markers in the sample to luminesce, and imaging the sample in an image frame via imaging optics along an imaging direction, wherein the image frame contains images of the luminescing fluorescence markers, and the imaging optics have a plane of focus and an optical resolution. The excitation step and imaging steps are repeated multiple times to generate a plurality of image frames, wherein the excitation steps are performed to isolate the images of the luminescing fluorescence markers in each image frame for at least some of the luminescing fluorescence markers. The location of the corresponding fluorescence marker is determined in each instance in the generated plurality of image frames from the isolated images of the luminescing fluorescence markers, and a highly resolved total image is generated from the locations determined in this way.

Method for three-dimensional high resolution localization microscopy

A method for SPIM microscopy, wherein the sample is moved continuously, and a plurality of images are taken at time intervals by means of a detection arrangement during the movement. The image capture duration or exposure time is dimensioned such that the movement path of the sample lies within a predetermined resolution range of the detection objective. The speed of the sample movement is determined and set by the image capture duration or exposure time and/or the distortion of the point spread function generated by the sample movement of the sample. The image blur is corrected computationally by the respective image capture duration and the movement speed. A sharp image is generated in this way. The actual optical section thickness of the light sheet is determined from the light sheet thickness, and the movement speed is determined therefrom and from user settings.

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Jena, Germany

Jörg Ritter