Two-color live-cell STED nanoscopy by click labeling with cell-permeable fluorophores

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are required. In addition, the fluorophores should preferentially emit in the red spectral range to reduce potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. Here we demonstrate two-color STED imaging of living cells using various pairs of dyes that fulfill all of the above requirements. To this end, we combine click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, enabling long-term STED imaging of different target structures in living specimens. ### Competing Interest Statement All authors declare no competing interests in the production and presentation of results. We note that authors: JR, FG, and CW work at Abberior which develops and manufactures fluorescent probes. AE & CW hold shares at Abberior Instruments which develops and manufactures super-resolution fluorescence microscopes, including the STEDYCON system used here.