Abstract
The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.
Highlights
Letter or super-resolved STED spot several times along a line, yielding intensity traces for each pixel along the line that are correlated to generate the final fluorescence correlation spectroscopy (FCS) data (correlation data G(τ) against correlation lag time τ) in confocal and STED separately. (B) In LIESS−FCS, confocal and super-resolved STED−FCS data are generated simultaneously by alternating confocal and STED modes in-between subsequent lines
Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy measures such dynamics with high spatial and temporal resolution
We demonstrate the potential of the LIESS− FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes
Summary
Extrapolated[9] (even in the case of more-advanced camerabased approaches)[10,11] because the relevant molecular scales are below the diffraction-limited spatial resolution of these techniques. FCS experiments have demonstrated mainly free homogeneous diffusion for DPPE and spatially distinct spots of slowed down diffusion in the case of SM, only visible in the STED recordings.[18] due to the lack of simultaneous information from confocal recordings (e.g., slow diffusion at the same locations), this observation using sSTED−FCS could not directly be attributed to trapping interactions as reported from single-point STED−FCS measurements.[14,22,23] Figure 3 shows representative LIESS−FCS data [correlation carpets in STED (dSTED = 100 nm) and confocal (dconf = 240 nm) modes as well as values of Drat over space] for DPPE (Figure 3A−C) and SM (Figure 3D−F). LIESS−FCS provides an unique tool for the investigation of the lateral organization of cellular membranes on variable length scales accounting for bias due to biological heterogeneity or photobleaching artifacts and for possibly answering long-standing questions of functional membrane heterogeneity.[1,2] a combination of LIESS−FCS with other spatiotemporal methodologies will undoubtedly provide invaluable insights into cellular dynamics in the future
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
