High-resolution scanning microscopy distinguishing between at least two spectral ranges

Abstract

For the purposes of high-resolution scanning microscopy, a sample (2) is excited by illumination radiation (5) to emit fluorescent radiation in such a way that the illumination radiation (5) is focused at a point in or on the sample (2), so as to form a diffraction-limited illumination spot (14). The point is imaged in a diffraction image (17) on a detector (19) in a diffraction-limited manner, wherein the detector (19) has detector elements (25) and a plurality of location channels (21) which resolve a diffraction structure of the diffraction image (17). The sample (2) is scanned with variousscanning positions with an increment smaller than half the diameter of the illumination spot (14). An image of the sample (2) with a resolution that is increased beyond a resolution limit of the image is generated from the data of the detector (19) and from the scanning positions associated with these data. In order to distinguish between at least two predetermined spectral channels (R, G) in thefluorescent radiation of the sample (2), for each location channel (21) there is an independent beam path leading to a separating element (30) that spectrally divides these beam paths into the spectral channels (R, G) and then remixes the spectral channels (R, G) of the different location channels into the same number, on additional independent beam paths (28), such that a plurality of the additional independent beam paths (28) receive the radiation in different spectral channels (R, G) and from different location channels (21), and each of these additional independent beam paths (28) leads to one of the detector elements (25).

Description

technical field [0001] The present invention relates to a method of high-resolution scanning microscopy of a sample, wherein the sample is excited by illuminating radiation to emit fluorescent radiation, wherein the illuminating radiation is focused to a point in or on the sample to form a diffraction-limited illumination spot, the A diffraction image of a spot imaged in a diffraction-limited manner onto a spatially resolved detector arrangement (which has a spatial resolution to resolve the diffractive structure of the diffraction image) that is displaced relative to the sample to different Intensity data is read from the detector arrangement for each scan position, and from this intensity data and the scan position assigned to it an image of the sample is produced with an increase beyond the The resolution of the resolution limit. [0002] The invention also relates to a microscope for high-resolution scanning microscopy, comprising a sample space that receives a sample tha...

AI Technical Summary

Problems solved by technology

However, it has the disadvantage that each color channel requires its own color separator and detector

This problem gets worse as the number of colors increases

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