Whole-Cell, 3D, and Multicolor STED Imaging with Exchangeable Fluorophores

We demonstrate stimulated
emission depletion (STED) microscopy
of whole bacterial and eukaryotic cells using fluorogenic labels that
reversibly bind to their target structure. A constant exchange of
labels guarantees the removal of photobleached fluorophores and their
replacement by intact fluorophores, thereby circumventing bleaching-related
limitations of STED super-resolution imaging. We achieve a constant
labeling density and demonstrate a fluorescence signal for long and
theoretically unlimited acquisition times. Using this concept, we
demonstrate whole-cell, 3D, multicolor, and live-cell STED microscopy.