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 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: 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: Methods and a device for imaging a sample stained with multiple dyes are disclosed. The methods enable nanoscopic imaging with up to molecular resolution to be placed in the spatial context of microscopic images, or nanoscopic tracking of individual molecules to be performed in the spatial context of a microscopic image. The methods combine different light-optical microscopy techniques in a particularly efficient manner. Molecular resolution can be achieved by a localization microscopic method, in particular by localization according to a MINFLUX or STED-MINFLUX method. These methods are characterized by the fact that, on the one hand, they are particularly gentle on the sample during the steps that precede imaging with molecular resolution, and on the other hand, they make optimal use of the fluorescence photons during localization or enable optimal use.

Abstract: The invention relates to a method for determining a reference time point (to) 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 (to) 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 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 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 invention relates to a method for punctiform illumination of a sample (1) in a microscope, more particularly a MINFLUX microscope, using illumination light, with the sample (1) being sequentially illuminated at the illumination points (3) of a predefined or predefinable illumination point pattern (2). The method is distinguished in that a lateral extent of the illumination point pattern (2) is smaller than the longest wavelength of the illumination light and in that the illumination points (3) are always illuminated exclusively with a time offset and in that a distinct individual light source (4) of a plurality thereof is assigned to each illumination point (3) of the 10 illumination point pattern (2) and each illumination point (3) is illuminated by the focus of an illumination light bundle (5) of the individual light source (4) assigned thereto.

Abstract: For setting a laser-scanning fluorescence microscope to a correct alignment in which an intensity maximum of excitation light and an intensity minimum of fluorescence inhibition light coincide in a focal area of an objective lens, a structure in a sample marked with a fluorescent dye is scanned with the intensity maximum of the excitation light to generate first and second pictures of the sample, the first picture corresponding to a higher and the second picture corresponding to a lower intensity of the fluorescence inhibition light. A spatial offset of a first image of the structure in the first picture with regard to a second image of the structure in the second picture is calculated; and the intensity maximum of the excitation light is shifted with regard to the intensity minimum of the fluorescence inhibition light in the direction of the offset calculated to set the microscope to the correct alignment.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: A high resolution laser scanning microscope has beam shaping elements configured to shape a beam of fluorescence inhibiting light which is directed into a back aperture of an objective connected to form an intensity minimum delimited by intensity maxima of the fluorescence inhibiting light in a focus of the objective. A plurality of optical elements including the objective and the beam shaping elements are arranged in a beam path of the beam to the focus. Using the microscope includes removing or exchanging or altering or adding at least one of the optical elements arranged in the beam path of the beam of fluorescence inhibiting light, and compensating a variation of polarization varying properties of the plurality of the optical elements, that is caused by removing or exchanging or altering or adding the at least one optical element, by adapting the beam shaping elements to the variation.

Abstract: For setting a laser-scanning fluorescence microscope to a correct alignment in which an intensity maximum of excitation light and an intensity minimum of fluorescence inhibition light coincide in a focal area of an objective lens, a structure in a sample marked with a fluorescent dye is scanned with the intensity maximum of the excitation light to generate first and second pictures of the sample, the first picture corresponding to a higher and the second picture corresponding to a lower intensity of the fluorescence inhibition light. A spatial offset of a first image of the structure in the first picture with regard to a second image of the structure in the second picture is calculated; and the intensity maximum of the excitation light is shifted with regard to the intensity minimum of the fluorescence inhibition light in the direction of the offset calculated to set the microscope to the correct alignment.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.

Abstract: Method of optical microscopy by scanning a sample containing an excitable species, the method comprising: directing a first and a second light beam onto respective, partially overlapped areas of the sample, wherein the first light beam is provided for exciting members of the excitable species, and the second light beam is provided for reducing the number of excited members; detecting an optical signal coming from the sample, comprising a main component and a spurious component, during consecutive first and second time gates, the first time gate being provided for detecting the optical signal for a time interval during which the main component and the spurious component are both present, and the second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero; processing the detected optical signal to separate its main component.

Abstract: A high resolution laser scanning microscope has beam shaping elements configured to shape a beam of fluorescence inhibiting light which is directed into a back aperture of an objective connected to form an intensity minimum delimited by intensity maxima of the fluorescence inhibiting light in a focus of the objective. A plurality of optical elements including the objective and the beam shaping elements are arranged in a beam path of the beam to the focus. Using the microscope includes removing or exchanging or altering or adding at least one of the optical elements arranged in the beam path of the beam of fluorescence inhibiting light, and compensating a variation of polarization varying properties of the plurality of the optical elements, that is caused by removing or exchanging or altering or adding the at least one optical element, by adapting the beam shaping elements to the variation.

Abstract: A device comprises two polarization-selective optical elements for separately modulating wave fronts of two components of a collimated light beam, which are transversally polarized in orthogonal directions. The two polarization-selective optical elements are first and second partial areas of one spatial light modulator (SLM) diffracting the light beam in backward direction. A mirror arranged between the first and second partial areas of the SLM reflects the light beam coming from the first partial area towards the second partial area. A wave plate arranged between the first partial area and the second partial area of the SLM rotates the polarization directions of both components of the light beam by 90°. The mirror reflects the first and second components of the light beam as parallel bundles of light rays resulting in a lateral offset between the first and second components of the light beam behind the second partial area of the SLM.

Abstract: A device comprises two polarization-selective optical elements for separately modulating wave fronts of two components of a collimated light beam, which are transversally polarized in orthogonal directions. The two polarization-selective optical elements are first and second partial areas of one spatial light modulator (SLM) diffracting the light beam in backward direction. A mirror arranged between the first and second partial areas of the SLM reflects the light beam coming from the first partial area towards the second partial area. A wave plate arranged between the first partial area and the second partial area of the SLM rotates the polarization directions of both components of the light beam by 90°. The mirror reflects the first and second components of the light beam as parallel bundles of light rays resulting in a lateral offset between the first and second components of the light beam behind the second partial area of the SLM.

Abstract: Method of optical microscopy by scanning a sample containing an excitable species, the method comprising: directing a first and a second light beam onto respective, partially overlapped areas of the sample, wherein the first light beam is provided for exciting members of the excitable species, and the second light beam is provided for reducing the number of excited members; detecting an optical signal coming from the sample, comprising a main component and a spurious component, during consecutive first and second time gates, the first time gate being provided for detecting the optical signal for a time interval during which the main component and the spurious component are both present, and the second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero; processing the detected optical signal to separate its main component.