Fluorescence scanning microscope and method for imaging sample

Abstract

A fluorescence scanning microscope is introduced, comprising an excitation light source which is designed to generate an excitation light distribution that excites fluorophores present in the sample in order to spontaneously emit fluorescence photons; a de-excitation light source which is designed to generate a de-excitation light distribution that de-excites the fluorophores excited by the excitation light distribution in the sample by means of a stimulated emission of fluorescence photons; an illumination unit which is designed to combine the excitation light distribution and the de-excitation light distribution in order to form a light distribution that scans over multiple illumination targets of the sample such that the intensity maximum of the excitation light distribution and the intensity minimum of the de-excitation light distribution spatially overlap in the respective illumination target; a detector which is designed to detect the fluorescence photons emitted from each illumination target (on the basis of the arrival time of the photons); and a processor. The processor is designed to analyze the arrival time of the fluorescence photons detected in respective illumination targets, to generate a first pixel and a second pixel on the basis of the analysis, the pixels representing respective illumination targets, to combine the first pixels in order to form a first sample image and the second pixels in order to form a second sample image, and to determine a spatial offset between the intensity maximum of the excitation light distribution and the intensity minimum of the de-excitation light distribution using the two sample images.

Description

technical field [0001] The present invention relates to a fluorescent scanning microscope and a method for imaging a sample using the fluorescent scanning microscope. Background technique [0002] In the field of fluorescence microscopy, for high-resolution imaging of samples, so-called STED methods are generally used, in which the sample is illuminated with a light distribution resulting from the superimposition of excitation light and de-excitation light. Here, STED stands for "Stimulated Emission Depletion". Here, the excitation light is intended to excite the fluorophores present in the sample so that they fluoresce spontaneously. In contrast, de-exciting light (whose wavelength is different from that of the excitation light) is used to de-excite the fluorophores excited by the excitation light through stimulated emission of fluorescence. In order to increase the image resolution, de-excitation light with a special light distribution is superimposed on the excitation l...

AI Technical Summary

Problems solved by technology

[0010] All of the above methods also have the disadvantage that the influence of the relative position of the sample on the excitation light and the de-excitation light cannot be considered

The disadvantage of this method is that, for optimization, multiple iterative steps are required, since this measure does not contain any information about the direction in which the de-excitation light distribution must be shifted in order to achieve an optimal superposition

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