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 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 present disclosure relates to a method for localizing or tracking individual emitters in a sample according to the MINFLUX principle, wherein the sample is illuminated at several illumination positions in a close range around the emitter from different illumination directions with modulation light which comprises a linearly or flatly extended intensity minimum. A position of the emitter in the sample is calculated from the emissions of the emitter detected at the illumination positions. The invention further relates to a light microscope for carrying out the method.

Abstract: The specification relates to a luminescence microscope comprising a first light source for generating an input light beam, a light modulator comprising a first active surface and a second active surface, for modulating the phase and/or amplitude of the light incident on the respective active surface, an objective lens for focusing the light into a sample so that an light intensity distribution is formed in the sample, wherein the luminescence microscope comprises a first beam displacement element for polarization-dependent generation of a first output light beam and/or a second output light beam forming an angle of less than 90°, wherein the first beam displacement element is arranged such that that the first output light beam impinges on the first active surface and the second output light beam impinges on the second active surface and a method for imaging a sample or for localizing or tracking emitters in the sample.

Abstract: The invention relates to a detection device (2) for a laser scanning microscope, the detection device (2) having a light inlet (4), at least one filter module (14) and at least one spatially resolving detector (22) and being configured to guide light from the light inlet (4) to the filter module (14) and from there to the spatially resolving detector (22), at least one filter module (14) being designed as a continuous filter module with two continuously tunable filter elements (16), and at least one compensator element (26) being arranged optically downstream of the continuous filter module (14), by means of which a focal position of light on the spatially resolving detector (22) can be adjusted.

Abstract: A method for localizing individual emitters in a sample with a first localization step and a second localization step with an increased accuracy including 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 a location correction compensating for the systematic deviation is applied to determine the illumination positions in the second localization step, as well as a light microscope and a computer program for carrying out the method.

Abstract: Scanning fluorescence microscopes and related methods are described for microscopically recording images of samples extending in three dimensions. In particular embodiments, the microscopes are configured for recording a first sectional image that is parallel to an optical axis of a microscope objective lens by scanning a sample with a focused excitation light distribution in a sectional area parallel to the optical axis of the microscope objective. The excitation light distribution may be corrected by a correction device according to initial adjustment values for adjustment parameters of an aberration correction function. The first sectional image is then evaluated and new adjustment values for the adjustment parameters are defined.

Abstract: The invention relates to a method for determining a reference time point (t0) by means of a light microscope (1), wherein a sample (2) is illuminated by first light pulses (P1) in order to excite light-emitting entities in the sample (2), wherein a light signal generated by means of the first light pulses (P1) is detected, and wherein the reference time point (t0) is determined based on the detected light signal, and wherein the light signal is detected in at least two measurements each in a first detection time window (G1), wherein a starting time point of the first detection time window (G1) is adjusted for each of the measurements. The invention further relates to a light microscope (1) and a computer program for carrying out the method.

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 invention is directed to a method for recording a motion trajectory of an individual particle in a sample and to a light microscope performing this method. Starting from an at least approximately known initial position, the particle is scanned with an intensity distribution of a scanning light comprising a local intensity minimum. When illuminated with the scanning light, the particle to be tracked generates a detectable light signal, from the intensity of which updated coordinates of the particle are calculated. According to the invention, the scanning is terminated when a second measured variable detected in parallel satisfies a termination criterion.

Abstract: A method for punctiform illumination of a sample with a MINFLUX microscope has the sample being sequentially illuminated at illumination points of a predefined or predefinable illumination point pattern. The lateral extent of the illumination point pattern is smaller than the longest wavelength of the illumination light. The illumination points are always illuminated exclusively with a time offset. A distinct individual light source is assigned to each illumination point of the illumination point pattern. Each illumination point is illuminated by the focus of an illumination light bundle of the individual light source.

Abstract: The present disclosure relates to method, computer programs with instructions, and apparatus for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample by means of a localization microscope. The present disclosure also relates to localization microscopes using such an apparatus. Light distributions arising due to interference of coherent light are used for determining the positions of the molecules. In the method, a plurality of light distributions are generated (S1) using a first light modulator having a plurality of switchable pixels. The first light modulator is arranged in an image plane of the localization microscope. Each light distribution has a local intensity minimum and regions with an intensity increase adjacent thereto. Each of the two or more molecules is illuminated (S2) with one light distribution. For each of the light distributions, photons emitted by the molecules are detected (S4) for different positionings of the light distribution.

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: An auxiliary apparatus for testing the confocality of a scanning and descanning microscope component group has a connector configured for connecting the auxiliary apparatus in a defined relative position to the scanning and descanning microscope component group, and an optical axis running at a fixed orientation with respect to the connector. Further, the auxiliary apparatus has an auxiliary detector with a plurality of auxiliary detection apertures in a plurality of auxiliary detection aperture positions that are arranged at distances in direction of the optical axis and laterally with respect to the optical axis; and an auxiliary light source providing auxiliary light through a plurality of auxiliary emission apertures in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis and laterally with respect to the optical axis.

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: 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: A bandpass filter for light has variable lower and upper cut-off wavelengths. The bandpass filter comprises an areal long-pass filter defining the variable lower cut-off wavelength and an areal short-pass filter defining the variable upper cut-off wavelength. The long-pass filter has different lower cut-off wavelengths in different first area regions which follow to one another in a first direction, and the short-pass filter has different upper cut-off wavelengths in different second area regions which follow to one another in a second direction. The long-pass filter and the short-pass filter are connected in series and spatially fixed relative to one another. The first direction and the second direction are oriented crosswise to one another.