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

荧光漂白是所有类型的超分辨率显微镜的基本挑战。在STED显微镜的背景下，这一挑战得到了解决，例如，通过开发巧妙的成像方案，将激发光减少到最低限度，或者通过开发新的和特别光稳定的荧光团。我们的假设是，可交换的荧光团是一个强大的，替代工具，实现“漂白独立”STED显微镜。我们的目标是发展这一概念，并建立可交换的荧光团，以成像固定和活细胞中的细胞蛋白质。我们将把这一概念整合到细胞显微镜下的蛋白质标记的重要方法中，即抗体染色和用蛋白质标签原位标记蛋白质。我们建议建立“漂白独立”STED显微镜，通过使用荧光团可逆地结合到他们的目标结构和交换与（大）水库，这导致更换荧光团成像，并最终光漂白。该概念需要荧光团的快速交换动力学、未漂白荧光团的足够大的储库，并且必须同时提供靶结构的高密度标记。这将显著扩展STED显微镜对全细胞3D、多色和长期活细胞成像的适用性。

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.