HIGH-RESOLUTION SCANNING MICROSCOPY WITH DISCRIMINATION BETWEEN AT LEAST TWO WAVELENGTH RANGES

摘要

For the purposes of high-resolution scanning microscopy, a sample (2) is excited by illumination radiation (5) to emit fluorescence radiation in such a way that the illumination radiation (5) is focused at a point in or on the sample (2) to form a diffraction-limited illumination spot (14). The point is imaged in a diffraction image (17) on a spatially resolving two-dimensional detector (19) in a diffraction-limited fashion, wherein the two-dimensional detector (19) has a spatial resolution which resolves a diffraction structure of the diffraction image (17). The sample (2) is scanned by means of various scanning positions with an increment that is 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 two-dimensional detector (19) and from the scanning positions assigned to these data. For the purposes of discriminating between at least two predetermined wavelength ranges in the fluorescence radiation of the sample (2), a number of Airy disks (30-33) corresponding to the at least two predetermined wavelength ranges are generated on the two-dimensional detector (19), which Airy disks are offset laterally from one another such that the diffraction image (17) consists of the mutually offset Airy disks (30-33). The Airy disks (30-33) are evaluated when generating the image of the sample (2).