Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses
2016
Authors: Sidenstein SC, D'Este E, Böhm MJ, Danzl JG, Belov VN, Hell SW
CellNetworks People: Hell Stefan
Journal: Sci Rep. 2016 May 25;6:26725. 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.