title: Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins.
abstract: We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzylguanine-substituted organic dyes, for labeling. Tetramethylrhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany.
authors.forename: Birka
authors.initials: B
authors.lastname: Hein
authors.forename: Katrin I
authors.initials: KI
authors.lastname: Willig
authors.forename: Christian A
authors.initials: CA
authors.lastname: Wurm
authors.forename: Volker
authors.initials: V
authors.lastname: Westphal
authors.forename: Stefan
authors.initials: S
authors.lastname: Jakobs
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
mesh_terms: D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D007089:Image Enhancement / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins