Light microscopy, allowing sub-diffraction-limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the reso-lution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the ﬁrst choice to obtain ultraresolution structures, brings highly speciﬁc labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of ﬂuorescence at cryogenic temperatures can contribute for ﬂuorescence imaging of biological samples, like whole cells, is described.
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- single molecules