Abberior Instruments GmbH

The present disclosure relates to an illumination and imaging method for light microscopy, wherein the illumination takes place at different axial positions in a sample, or parts of the sample are imaged from different depths. The present disclosure further relates to localization methods for individual emitters in a sample using such an illumination method, in particular to localization methods according to the MINFLUX principle. Finally, the present disclosure comprises a light microscope for carrying out an illumination, imaging and/or localization method according to the present disclosure.

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 arranged around the position of the emitter determined in the preceding iteration, wherein the illumination positions in the first iteration and the at least one second iteration form a respective illumination pattern comprising a maximum extension, wherein the maximum extension of the illumination pattern is kept constant during the main localization. The invention further relates to a light microscope for carrying out the method, a computer program and the use of a fiber bundle for the method.

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.

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.

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.

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.

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. Further image data are then recorded by scanning the sample with the focused excitation light distributions, with the excitation light distribution corrected by the correction device according to the new adjustment values for the adjustment parameters of the aberration correction function.

The invention relates to a method for determining a reference time point (t 0 ) by means of a light microscope ( 1 ), wherein a sample ( 2 ) is illuminated by first light pulses (P 1 ) in order to excite light-emitting entities in the sample ( 2 ), wherein a light signal generated by means of the first light pulses (P 1 ) is detected, and wherein the reference time point (t 0 ) 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 (G 1 ), wherein a starting time point of the first detection time window (G 1 ) 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.

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.

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.