Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses

• Published in: Scientific reports Vol. 6; no. 1; p. 26725
• Main Authors: Sidenstein, Sven C., D’Este, Elisa, Böhm, Marvin J., Danzl, Johann G., Belov, Vladimir N., Hell, Stefan W.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.05.2016; Nature Publishing Group
• Subjects: 631/1647/328/2238; 639/624/1107/328/2238; Actin; Actins - metabolism; Animals; Cytoskeleton; Dendritic spines; Female; Fluorophores; HeLa Cells; Hippocampus; Hippocampus - cytology; Hippocampus - metabolism; Humanities and Social Sciences; Humans; Male; Microscopy; Molecular Imaging - methods; multidisciplinary; Neurons - cytology; Neurons - metabolism; Rats; Rats, Wistar; Science; Science (multidisciplinary); Spectrin; Spectrin - metabolism; Synapses - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep26725

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.