High-resolution luminescence microscopy
Abstract:
A microscopy method for generating a high-resolution image (55) of a sample (2) comprising the following steps: a) the sample (2) is provided with a marker, which upon excitation emits statistically flashing luminescent radiation, or a sample (2) is used which upon excitation emits locally distributed, statistically flashing luminescent radiation; b) the sample (2) is excited to luminescence by means of structured illumination, wherein the sample is repeatedly illuminated in at least nine different illumination conditions (0.01-0.09) of the structured illumination by realizing at least three rotary positions, and at least three displacement positions per rotary position of the structured illumination; c) the luminescing sample (2) is repeatedly displayed in each of the different illumination conditions on a flat panel detector having pixels, such that an image sequence (44.01-44.09) is obtained for each of the different illumination conditions (0.01-0.09); d) a raw image, the spatial resolution of which exceeds the optical resolution of the image is generated from each image sequence (44.01-44.09) by means of a cumulant function (46), which evaluates intensity fluctuations of each pixel in the image sequence, said intensity fluctuations being caused by the flashing, so that for each of the different illumination conditions (0.01-0.09), a high-resolution raw image (53.01-53.09) is obtained; e) from the raw images (53.01-53.09) thus obtained, the high-resolution image (55), which relative to the raw images (53.01-53.09) has an increased spatial resolution, is generated by way of computational processing (42) comprising Fourier filtering.
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