Method and apparatus for the localization of a single dye molecules in fluorescence microscopy

摘要

The invention concerns a method and a corresponding apparatus for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The method comprises: - acquiring localization image data by employing fluorescence localization microscopy, wherein said localization image data comprises a series of images obtained by - illuminating a region of interest of the sample with illumination light having intensity in the range of approximately 1 kW/cm2 to approximately 1 MW/cm2, - detecting by an information acquiring sensor at least a portion of the fluorescent light emitted by at least a portion of the fluorescent molecules of the at least one type fluorescent label upon illumination, thereby obtaining an image of the region of interest; - repeating the steps of illuminating and detecting of the emitted fluorescent light a plurality of times, thereby obtaining the series of images, each image being taken at a different time step; and - processing the acquired localization image data to thereby obtain said localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, wherein the step of processing comprises determining in each of the detected images of the series the positions of the barycenters of the detected fluorescence emission distributions from the single fluorescent molecules of the one or more fluorescent labels in at least one spatial direction.