Carl Zeiss Microscopy Gmbh

Lutz Schaefer

Ingo Kleppe

Yauheni Novikau

Markus Sticker

Christoph Husemann

A method for determining the optimal position of the focal plane for examining a specimen by microscopy can include a) illuminating the specimen with light and recording images at different positions of the focal plane to provide a stack of intensity images, b) calculating a phase image from at least two intensity images, with the calculated phase image being assigned a focal plane position located within a focal plane region whose boundaries are the two most spaced apart positions of the focal plane of the at least two intensity images, c) repeating step b) multiple times with different intensity images such that a stack of phase images is available, d) calculating at least one focus measure value for each phase image, and e) determining the optimal position of the focal plane on the basis of the calculated focus measure values and the focal plane positions assigned to the phase images.

Method and device for determining the optimal position of the focal plane for examining a specimen by microscopy

A method of image evaluation when performing SIM microscopy on a sample includes: A) providing n raw images of the sample, which were each generated by illuminating the sample with an individually positioned SIM illumination pattern and imaging the sample in accordance with a point spread function, B) providing (S) n illumination pattern functions, which each describe one of the individually positioned SIM illumination patterns, C) providing (S) the point spread function and D) Carrying out an iteration method, which includes following iteration steps a) to e), as follows: a) providing an estimated image of the sample, b) generating simulated raw images, in each case by image processing of the estimated image using the point spread function and one of the n illumination pattern functions such that n simulated raw images are obtained, c) assigning each of the n simulated raw images to that of the n provided raw images which was generated by the illumination pattern that corresponds to the illumination pattern function used to generate the simulated raw image, and calculating n correction raw images by the comparison of each provided raw image with the simulated raw image assigned thereto, d) generating a correction image by combining image processing of the n correction raw images using the point spread function and the n illumination pattern functions, wherein a filtering step is carried out in each implementation of iteration step d), said filtering step suppressing a spatial fundamental frequency of the illumination pattern, and e) reconstructing the estimated image of the sample by means of the correction image and using the corrected estimated image of the sample as the estimated image of the sample in iteration step a) in the next run through the iteration.

Method of image evaluation for sim microscopy and sim microscopy method

A method for determining the position of an optical boundary surface along a first direction includes a) imaging a pattern in a plane transverse to the first direction, and recording a two-dimensional image of the pattern imaged in the plane; b) repeating step a) for different positions in the first direction, wherein the different positions cover an area in the first direction in which the optical boundary surface lies; c) averaging each image from step a) along a direction transverse to the second direction such that in each case a one-dimensional data set is produced; d) transforming each data set from step c) into a frequency domain; e) determining the frequency of the greatest amplitude of all data sets transformed in step d); and f) determining the position along the first direction by evaluating the data sets transformed in step d) at the frequency determined in step e).

Growing community of inventors

Kitchener, Canada

Lutz Schaefer