title: STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics.
abstract: Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.
authors.affiliation: Nanoscopy, Nanophysics, Instituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.|Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Vicidomini
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.
authors.forename: Haisen
authors.initials: H
authors.lastname: Ta
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.|Max Planck Institute for Molecular Cell Biology and Genetics , Pfotenhauerstr. 108, 01309 Dresden, Germany.
authors.forename: Alf
authors.initials: A
authors.lastname: Honigmann
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mueller
authors.affiliation: MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Molecular Medicine, University of Oxford , OX3 9DS Oxford, United Kingdom.|MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark , Campusvej 55, Odense MDK-5230, Denmark.
authors.forename: Mathias P
authors.initials: MP
authors.lastname: Clausen
authors.affiliation: MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Molecular Medicine, University of Oxford , OX3 9DS Oxford, United Kingdom.
authors.forename: Dominic
authors.initials: D
authors.lastname: Waithe
authors.affiliation: MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Molecular Medicine, University of Oxford , OX3 9DS Oxford, United Kingdom.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galiani
authors.affiliation: MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Molecular Medicine, University of Oxford , OX3 9DS Oxford, United Kingdom.
authors.forename: Erdinc
authors.initials: E
authors.lastname: Sezgin
authors.affiliation: Nanoscopy, Nanophysics, Instituto Italiano di Tecnologia , Via Morego 30, 16163 Genoa, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Goettingen, Germany.|MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Molecular Medicine, University of Oxford , OX3 9DS Oxford, United Kingdom.
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D008563:Membrane Lipids / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D056004:Molecular Dynamics Simulation; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008563:Membrane Lipids
keywords: Super-resolved microscopy; fluorescence-correlation spectroscopy; stimulated-emission-depletion microscopy; time-correlated single-photon counting; time-resolved