Two-colour live-cell nanoscale imaging of intracellular targets

Authors:

Bottanelli, F., Kromann, E. B., Allgeyer, E. S., Erdmann, R. S., Baguley, S. W., Sirinakis, G., ... & Bewersdorf, J.

Abstract:

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.

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