Multicolour multilevel STED nanoscopy of actin/spectrin organization at synapses Journal Article

Author(s): Sidenstein, Sven C; D'Este, Elisa; Böhm, Marvin J; Danzl, Johannes G; Belov, Vladimir N; Hell, Stefan W
Article 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 ("multilevelSTEDv) 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.
Journal Title: Scientific Reports
Volume: 6
ISSN: 20452322
Publisher: Nature Publishing Group  
Date Published: 2016-05-25
Start Page: 1
End Page: 8
Copyright Statement: CC BY
DOI: 10.1038/srep26725
Notes: We acknowledge the assistance of I. Herfort with neuron preparation, and of J. Bienert and K. Müller with analyses of the dye 540R derivatives. We thank T. Gilat and E. Rothermel for sample preparation as well as J. Keller, F. Winter (all MPI-BPC) and C.A. Wurm (Abberior Instruments) for helpful discussion, and S.J. Sahl (MPI-BPC) for a critical reading of the manuscript.
Open access: yes (OA journal)