Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

Nature Protocols
Erdinc SezginChristian Eggeling


Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.


Jul 19, 2000·Proceedings of the National Academy of Sciences of the United States of America·T A KlarS W Hell
Dec 7, 2002·Nature Biotechnology·Antje KepplerKai Johnsson
Dec 5, 2003·Journal of the American Chemical Society·Susanne GendreizigKai Johnsson
Apr 14, 2004·Current Pharmaceutical Biotechnology·Jörg EnderleinJörg Fitter
May 21, 2005·Physical Review Letters·Lars KastrupStefan W Hell
Oct 4, 2005·Biophysical Journal·Laure WawrezinieckPierre-François Lenne
Dec 22, 2005·Biochimica Et Biophysica Acta·Akihiro Kusumi, Kenichi Suzuki
Apr 15, 2006·Science·Ben N G GiepmansRoger Y Tsien
Jun 6, 2006·Biophysical Journal·Jana HumpolíckováYves Engelborghs
Jul 22, 2006·The EMBO Journal·Pierre-François LenneDidier Marguet
Nov 7, 2006·Biophysical Journal·Jérôme WengerPierre-François Lenne
Nov 1, 2007·Nature Methods·Sally A KimPetra Schwille
Nov 17, 2007·Nature Protocols·Kirsten Bacia, Petra Schwille
Dec 13, 2007·Optics Letters·T A Klar, S W Hell
Feb 23, 2008·Chemistry & Biology·Arnaud GautierKai Johnsson
Jun 7, 2008·ACS Chemical Biology·Georgyi V LosKeith V Wood
Jul 22, 2009·Biophysical Journal·Michelle A Digman, Enrico Gratton
Aug 8, 2009·Biochemistry·Rita L StrackBenjamin S Glick
Feb 23, 2010·Optics Express·Gael MoneronStefan W Hell
Mar 24, 2010·Journal of the American Chemical Society·Jun-Ichi HottaJohan Hofkens
Jul 21, 2010·Biophysical Journal·Kateryna S MorozovaVladislav V Verkhusha
Oct 30, 2010·Nature Protocols·Molly J RossowEnrico Gratton
Mar 4, 2011·Optics Express·Jeffrey R MoffittJens Michaelis
Jun 22, 2011·Biophysical Journal·Brian R RankinStefan W Hell
Jan 3, 2012·Biophysical Journal·Elliot L Elson
Mar 28, 2012·Biochimica Et Biophysica Acta·Erdinc SezginPetra Schwille
Mar 29, 2012·Optics Express·Silvia GalianiPaolo Bianchini
Jun 30, 2012·Nature Methods·Johannes SchindelinAlbert Cardona
Jul 10, 2013·Proceedings of the National Academy of Sciences of the United States of America·Carmine Di RienzoFrancesco Cardarelli
Oct 11, 2013·Methods in Molecular Biology·Tomáš SteinbergerMartin Hof
Nov 10, 2013·PloS One·Franziska StaggeStefan Jakobs
May 16, 2014·Bioinformatics·Paul MüllerThomas Weidemann
Jun 27, 2014·PloS One·Paolo BianchiniRanieri Bizzarri
Aug 26, 2014·ACS Nano·John T KingSteve Granick
Sep 30, 2014·Nature Methods·Patrick TheerMichael Knop

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Methods Mentioned

imaging techniques
fluorescence microscopy
optical microscopy
super-resolution microscopy

Software Mentioned

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