Bleaching-independent, multi-color, whole-cell STED microscopy using exchangeable fluorophores

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Fluorophore bleaching is a fundamental challenge in all types of super-resolution microscopy. In the context of STED microscopy, this challenge was addressed, for example, by developing clever imaging schemes that reduce the excitation light to a minimum, or through the development of new and exceptionally photostable fluorophores. Our hypothesis is that exchangeable fluorophores are a powerful, alternative tool to realize “bleaching-independent” STED microscopy. We aim to develop this concept and establish exchangeable fluorophores to image cellular proteins in fixed and live cells. We will integrate this concept into important methods of protein labeling for microscopy of cells, i.e. antibody staining and in situ protein labeling with protein tags. We propose to establish "bleaching-independent" STED microscopy by using fluorophores that reversibly bind to their target structure and exchange with a (large) reservoir, which leads to a replacement of fluorophores that were imaged and eventually photobleached. This concept requires a fast exchange kinetics of the fluorophores, a sufficiently large reservoir of unbleached fluorophores, and must at the same time provide a high-density labeling of a target structure. This will significantly extend the applicability of STED microscopy towards whole-cell 3D, multi-color and long-term live cell imaging.