title: Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses.
abstract: Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels ("multilevelSTED") optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Sven C
authors.initials: SC
authors.lastname: Sidenstein
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: D'Este
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Marvin J
authors.initials: MJ
authors.lastname: Böhm
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Johann G
authors.initials: JG
authors.lastname: Danzl
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Belov
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D005260:Female; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008297:Male; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013049:Spectrin / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*
chemical_list: D000199:Actins; D013049:Spectrin