High-resolution scanning microscopy resolving at least two spectral ranges

Abstract

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