Two-colour live-cell nanoscale imaging of intracellular targets

• Published in: Nature communications Vol. 7; no. 1; p. 10778
• Main Authors: Bottanelli, Francesca, Kromann, Emil B., Allgeyer, Edward S., Erdmann, Roman S., Wood Baguley, Stephanie, Sirinakis, George, Schepartz, Alanna, Baddeley, David, Toomre, Derek K., Rothman, James E., Bewersdorf, Joerg
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04-03-2016; Nature Publishing Group; Nature Portfolio
• Subjects: 14/19; 14/34; 14/63; 631/57/2282; 631/80/2373; Animals; Chlorocebus aethiops; COS Cells; HeLa Cells; Heterocyclic Compounds, 4 or More Rings - chemistry; Humanities and Social Sciences; Humans; Luminescent Proteins; Microscopy, Fluorescence - methods; multidisciplinary; Nanotechnology - methods; Rhodamines - chemistry; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms10778

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.