TIME-RESOLVED IMAGING METHOD WITH HIGH SPATIAL RESOLUTION

摘要

Method for operating a point laser- scanning microscope, comprising scanning a sample with a focused illumination laser beam (EB); recording, by means of an array of detecting elements (23), a plurality of images of the sample over a scan by the laser beam, wherein the detecting elements (23) are configurable to an intensity mode, in which the recorded images are intensity images g_i,j(n) related to photons collected during an entire dwell time of the illumination beam on the individual position n, or in a time-resolved mode, in which the recorded images are time -resolved images g^t _ij(n, t), in which the collected photons are discriminated based on their times of arrival to the individual detecting elements (23); calculating a fingerprint image a by summing the plurality of intensity images g_i,j(n) over all positions n of the illumination beam (EB) on the sample, the fingerprint image depending simultaneously on an illumination point-spread function, hereinafter illumination PSF, h^exc, and a detection point-spread function, hereinafter detection PSF, h^det, estimating shift matrices s^x and s^y from the intensity images g_i,j(n), reconstructing at least one of: i) a time-resolved object function f^t, and ii) an intensity object function f, and, visualizing at least one of a high-resolution time-resolved image f^t~ and a high- resolution intensity image f^~, based on said time -resolved object function and intensity object function.