title: Two-colour live-cell nanoscale imaging of intracellular targets.
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.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bottanelli
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Emil B
authors.initials: EB
authors.lastname: Kromann
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Edward S
authors.initials: ES
authors.lastname: Allgeyer
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Erdmann
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wood Baguley
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
authors.forename: George
authors.initials: G
authors.lastname: Sirinakis
authors.affiliation: Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.|Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Nanobiology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baddeley
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Nanobiology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Derek K
authors.initials: DK
authors.lastname: Toomre
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Nanobiology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Rothman
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06520, USA.|Nanobiology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Joerg
authors.initials: J
authors.lastname: Bewersdorf
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry*
chemical_list: C521550:Atto590; D006576:Heterocyclic Compounds, 4 or More Rings; D008164:Luminescent Proteins; D012235:Rhodamines