Abberior Instruments Gmbh

Roman Schmidt

Lars Kastrup

Benjamin Harke

Gerald Donnert

Matthias Reuss

Winfried Willemer

Andreas Schönle

The present disclosure relates to MINFLUX nanoscopy. The present disclosure improves three-dimensional localization of isolated emitters, particularly of isolated fluorescent emitters. The utilization of the emitted photons of isolated excitable emitters, in particular of isolated excitable fluorescent emitters, for a three-dimensional localization is improved by separating the localization according to the MINFLUX principle using a 3D excitation donut into a sequence of separate steps, wherein in one step either an axial location or a lateral location of the fluorophore is determined. Thereby, the more precise knowledge of the axial position increases the quality of a following lateral localization and vice versa. The three-dimensional localization is further improved by a method for axial localization by scanning an axial scanning area with a 3D excitation donut and a preferably following evaluation of the measured intensities or photon numbers by forming a vector sum.

Method and apparatus for high-resolution localization of a single emitter in multiple spatial directions

The invention relates to a method for localizing an emitter (F) in a sample (S) comprising illuminating the sample (S) with a stationary donut-shaped excitation beam (E), acquiring fluorescence photons; and estimating a position of the emitter (F) in the sample (S) from the acquired fluorescence photons. The invention further relates to an apparatus () for localizing an emitter (F) in a sample (S) comprising illumination means (), acquisition means () and processing means () and a computer program comprising instruction to cause the apparatus () to execute the method for localizing an emitter (F).

Method, apparatus and computer program for localizing an emitter in a sample

The present disclosure is directed to a method of disturbance correction and to a laser scanning microscope carrying out this method. Specifically, it is directed to an image recording method according to the MINFLUX principle, in which a spatially isolated fluorescence dye molecule is illuminated at a sequence of scan positions by an intensity distribution with a local intensity minimum, and the number of fluorescence photons emitted by the fluorescence dye molecule is detected at each of the scan positions. The location of the molecule is determined with a high spatial resolution from the scan positions and the numbers of fluorescence photons. A disturbance is captured when illuminating the fluorescence dye molecule and detecting the fluorescence light, said disturbance being considered in corrective fashion when determining the location of the fluorescence dye molecule.

Method of disturbance correction, and laser scanning microscope having disturbance correction

The present invention is related to a method, a computer program, and apparatus for adapting an estimator for use in a microscope for estimating a position of an emitter in a sample based on a method, in which the sample is illuminated with light at one or more sets of probe positions and fluorescence photons are acquired for the sets of probe positions. The invention is further related to a microscope, which makes use of such a method or apparatus. The sample is illuminated with light at one or more sets of probe positions and fluorescence photons are acquired for the sets of probe positions. Photon counts of the acquired photons are then added to vectors of photon counts or sums of photon counts are determined for the sets of probe positions. A value representative of background noise is determined and used for adapting the estimator in real-time.

Method, computer program, and apparatus for adapting an estimator for use in a microscope

An apparatus for detecting movements of a sample with respect to an objective includes imaging optics which include the objective, which have an image plane and which are configured to image light from at least one reference object that is connected to the sample arranged in front of the objective into reference object images in the image plane. The apparatus further includes a camera which is arranged in the image plane of the imaging optics and which is configured to record the reference object images at consecutive points in time, and an optical device arranged between the objective and the camera in a plane that is Fourier-conjugated with respect to the image plane. The optical device is configured to mask out low spatial frequencies of reference object images which the imaging optics image into the image plane.

Detecting movements of a sample with respect to an objective

The present specification relates to a method for light microscopic examination of a sample (), in particular by means of laser scanning or MINFLUX microscopy, in which a drift of the sample () or of an object in a sample () with respect to the light microscope () is detected and, if necessary, corrected. In particular, the present specification relates to a corresponding method for examining the sample () using laser scanning or MINFLUX microscopy. For this purpose, reference markers () are located in the sample, the position of which is repeatedly determined according to the MINFLUX principle in order to determine the drift.

Method and light microscope for a high-resolution examination of a sample

For detecting movements of a sample with respect to an objective, the sample is imaged onto an image sensor comprising an array of pixels by means of the objective. Images of the sample are recorded in that light coming from the sample is registered at the pixels. Variations of intensities of the light coming from the sample and registered at the pixels are determined during a set-up period in that a temporal course of the intensity of the light, which has been registered at a respective one of the pixels over the set-up period, is analyzed. Using these variations as a criterion, a subset of not more than 90% of the pixels of the image sensor is selected. Parts of the images that each correspond to the selected subset are compared to parts of at least one reference image that also correspond to the subset.

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Goettingen, Germany

Roman Schmidt