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.
Highlights
A widespread tool for investigating molecular diffusion dynamics is fluorescence correlation spectroscopy (FCS), which determines average molecular diffusion coefficients D from thousands of molecular transits through the microscope’s observation spot.[6,7] With measurement times of only a few seconds, and by placing or scanning the spot over distinct points in space, FCS may deliver information on heterogeneity in diffusion over space and time.[8,9] Revealing heterogeneous diffusion modes requires probing diffusion coefficients D at different spatial scales, i.e., for different observation spot diameters d5,10 (Figure 1b)
stimulated emission depletion (STED)-fluorescencelifetime-correlation spectroscopy (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
A remedy to this limitation is the use of FCS on a superresolution stimulated emission depletion (STED) micro
Summary
STED-FLCS An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics Vicidomini, Giuseppe; Ta, Haisen; Honigmann, Alf; Mueller, Veronika; Clausen, Mathias P.; Waithe, Dominic; Galiani, Silvia; Sezgin, Erdinc; Diaspro, Alberto; Hell, Stefan W.; Eggeling, Christian. P., Waithe, D., Galiani, S., Sezgin, E., Diaspro, A., Hell, S. Terms of use This work is brought to you by the University of Southern Denmark. If no other license is stated, these terms apply:. Giuseppe Vicidomini,*,†,‡ Haisen Ta,‡ Alf Honigmann,‡,§ Veronika Mueller,‡ Mathias P. Clausen,∥,⊥ Dominic Waithe,∥ Silvia Galiani,∥ Erdinc Sezgin,∥ Alberto Diaspro,† Stefan W.
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