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Two-colour live-cell nanoscale imaging of intracellular targets.

  • Author(s): 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
  • et al.
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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