Abstract
AbstractThis chapter provides a comprehensive overview of how reliable structural information can be obtained from super-resolution imaging based on stochastic photoswitching of organic fluorophores in fixed and living cells. Since single-molecule-based super-resolution imaging relies critically on the labeling density, the reversibility of photoswitching, and exact fitting of the center of mass of the measured point spread functions (PSFs) of isolated fluorophores, the controlled photoswitching of organic fluorophores with minimal photobleaching are discussed in detail, with particular focus on how they influence structural information extractable. Furthermore, the mechanism of reversible photoswitching of organic fluorophores in aqueous solvents in the presence of thiols is described. Finally, representative applications of direct stochastic optical reconstruction microscopy (dSTORM) are provided and consequences for live-cell super-resolution imaging with organic fluorophores with high spatiotemporal resolution is discussed.Keywords dSTORMNanoscopyOrganic fluorophoresPhotoswitchingPhotophysicsSuper-resolution imaging
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