Stephanie Wood Baguley, Edward S. Allgeyer, Alanna Schepartz, David Baddeley, George Sirinakis, Roman S. Erdmann, Emil B. Kromann, Francesca Bottanelli, James E. Rothman, Joerg Bewersdorf, Derek Toomre, Allgeyer, Edward [0000-0002-2187-4423], Sirinakis, George [0000-0002-4762-422X], and Apollo - University of Cambridge Repository
Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution., The intracellular applications of STED microscopy are limited by the availability of dyes. Here the authors develop a two-colour labelling strategy based on SiR and ATTO590 dyes, and apply their strategy to image various subcellular membrane compartments.