Abstract: The present disclosure relates to a scanning light microscopy method comprising illuminating a sample with illumination light, scanning a focused light distribution of the illumination light over or through the sample by means of a scanner (4) comprising adjustable control parameters, and detecting light emitted by light-emitting entities in the sample in response to the illumination light to obtain an image of the sample or to obtain a localization map of single light-emitting entities in the sample, wherein the method is performed by a scanning light microscope (1) which is operable in at least two different operation modes, and wherein the method comprises adjusting the control parameters of the scanner (4) dependent on the operation mode, in which the scanning light microscope (1) is operated. The present disclosure further relates to a scanning light microscope (1) and a computer program for performing the method.

Abstract: The present specification relates to a method for localizing or tracking emitters in a sample, wherein the sample is illuminated with an intensity distribution of an illumination light comprising a local minimum, wherein light emissions of a measuring emitter induced or modulated by the illumination light are detected, and wherein a position of the measuring emitter is estimated based on the detected light emissions and assigned positions of the local minimum, wherein the estimated position of the measurement emitter is corrected based on calibration data dependent on a speed and/or an acceleration of a measurement scanning movement of a scanning device, or wherein an actuation signal of the scanning device is adapted based on calibration data, wherein the calibration data comprise localization data of a calibration emitter obtained by means of at least one calibration scanning movement of the scanning device.

Abstract: 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.

Abstract: The disclosure relates to a method and a microscope for localizing and tracking singulated emitters in a sample. A sample is exposed to an intensity distribution of an illumination light comprising a local intensity minimum in a close region of the singulated emitter. The position of the intensity distribution in the sample fluctuates around a nominal position. The sample is excited to emit and the emissions are detected. Measured variables are recorded in such a way that the current positions of the intensity distribution in the sample can be assigned to the detected emissions. A position of the emitter can be estimated from the detected emissions and the assigned current positions of the intensity distribution.

Abstract: 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.

Abstract: 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.

Abstract: The present specification relates to a method for light microscopic examination of a sample (6), in particular by means of laser scanning or MINFLUX microscopy, in which a drift of the sample (6) or of an object in a sample (6) with respect to the light microscope (26) is detected and, if necessary, corrected. In particular, the present specification relates to a corresponding method for examining the sample (6) using laser scanning or MINFLUX microscopy. For this purpose, reference markers (8, 13) are located in the sample, the position of which is repeatedly determined according to the MINFLUX principle in order to determine the drift.

Abstract: 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.

Abstract: The invention relates to a method for determining a position of at least one emitter in a sample, wherein according to a measuring parameter, the sample is illuminated by activation light, the sample is illuminated by an excitation light intensity distribution comprising a local minimum flanked by maxima, the excitation light intensity distribution is positioned at a plurality of probing positions determined based on an initial position value, an emission signal from the at least one emitter is detected for each of the probing positions, at least one final position value is determined from the emission signals and the associated probing positions, and a position of the emitter is estimated based on the at least one final position value, wherein the measuring parameter is adjusted based on an adjusted speed parameter. The invention further relates to a device and a computer program for implementing the method.

Abstract: Methods, computer programs with instructions, and apparatus for estimating a position of an emitter or reflector in a sample are disclosed. Also disclosed are microscopes using a method or an apparatus according to the present principles. In the method, the sample is illuminated with excitation light at at least one set of target coordinates. Fluorescence photons or reflected photons are detected for the individual target coordinates of the set of target coordinates. A position of an emitter or reflector is estimated from the detected fluorescence photons or reflected photons. The estimation of the position of the emitter or reflector comprises a comparison of estimated positions determined from subsets of the set of target coordinates. Alternatively, the excitation light has an intensity distribution having a central minimum and three or more local maxima arranged around the central minimum, wherein the angular positions of the three or more local maxima change along the optical axis.

Abstract: 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.

Abstract: Embodiments of the invention relates to a method for localizing or tracking emitters in a sample, wherein the sample is illuminated with an intensity distribution of an illumination light having a local minimum, wherein the illumination light induces or modulates light emissions of the emitters, and wherein the local minimum is positioned in a region around a presumed position of an emitter in the sample, detecting light emissions (L) of the emitter, and determining the position of the emitter in the sample, wherein light emanating from the sample is detected with a plurality of detector elements having respective active areas whose projections into a focal plane in the sample are not congruent, wherein a background is estimated based on the light detected by the plurality of detector elements, and wherein a background correction is performed, a light microscope and a computer program for performing the method.

Abstract: 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.

Abstract: The present specification relates to a method for light microscopic examination of a sample (6), in particular by means of laser scanning or MINFLUX microscopy, in which a drift of the sample (6) or of an object in a sample (6) with respect to the light microscope (26) is detected and, if necessary, corrected. In particular, the present specification relates to a corresponding method for examining the sample (6) using laser scanning or MINFLUX microscopy. For this purpose, reference markers (8, 13) are located in the sample, the position of which is repeatedly determined according to the MINFLUX principle in order to determine the drift.

Abstract: The invention relates to a method for localizing individual emitters in a sample, comprising a pre-localization comprising an illumination of the sample with illumination light, wherein the illumination light induces or modulates light emissions of an individual and stationary emitter in the sample, detecting the light emissions of the emitter and estimating the position of the emitter in the sample from the detected light emissions and a subsequent main localization comprising illuminating the sample with an intensity distribution of the illumination light at illumination positions, the intensity distribution comprising a local minimum, detecting the light emissions of the emitter for the illumination positions, and determining the position of the emitter in the sample from the light emissions detected for the illumination positions, wherein the illumination positions are arranged in a first iteration about the estimated position and wherein the illumination positions in at least one second iteration are arr

Abstract: The invention relates to a method for localizing individual emitters in a sample with a first localization step comprising illuminating the sample with illumination light, detecting the light emissions of the emitter and determining the location of the emitter in the sample from the detected light emissions, and a second localization step with an increased accuracy compared to the first localization step comprising illuminating the emitter with an intensity distribution of the illumination light having a local minimum at illumination positions arranged around the location of the emitter determined in the first localization step, detecting the light emissions of the emitter for the illumination positions, and determining the location of the emitter from the light emissions detected for the illumination positions, wherein the location of the emitter determined in the first localization step comprises a systematic deviation with respect to the location of the same emitter determined in the second localization st

Abstract: The present invention is a method for spatially highly accurate location determination of individual dye molecules of a fluorescent dye by scanning with an intensity distribution of a scanning light having a local minimum. The invention is characterized by the fact that the scanning is not performed uniformly for all dye molecules, but is individually adapted to the dye molecule to be scanned and, if necessary, to its environment in the sample, in order to achieve the most accurate location determination possible with the smallest possible number of fluorescence photons.

Abstract: 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.

Abstract: The present disclosure relates to localisation microscopic investigations of samples stained with multiple dyes. According to the present disclosure, it is either provided that a singulated excitable fluorophore of a first species is excited with excitation light of two different wavelengths and that a localisation of the fluorophore is obtained for each of the two wavelengths, or that first test excitation is performed in order to then select a wavelength for excitation light with which a singulated fluorophore is localised. In the first case, the difference in the localisations of the one or preferably multiple individual fluorophores obtained in this way is determined and used to obtain localisations of fluorophores of a different species and those of the first species in a common spatial reference system. The second case is advantageously applicable for tracking structures. Also in the second case, the advantages of the first case can be additionally realised.

Abstract: The invention relates to a method for estimating a position of an emitter (2) in a sample (1) comprising illuminating the sample (1) with light at one or more sets of probe positions (Pi), acquiring photons for the sets of probe positions (Pi), and determining vectors of photon counts or sums of photon counts for the sets of probe positions from the acquired photons, and-estimating the position of the emitter (2) from the vectors of photon counts or sums of photon counts, wherein the one or more sets of probe positions (Pi) each comprise six or more probe positions (Pi), which are arranged rotationally symmetric on a circle around a center, wherein the one or more sets of probe positions lacks a central probe position. The invention further relates to an apparatus (40) for implementing the method, a microscope (10) using the apparatus (40) and a computer program implementing the method.