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Nano Lett. 2018 Jul 11;18(7):4233-4240. doi: 10.1021/acs.nanolett.8b01190. Epub 2018 Jun 19.

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging.

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Research Complex at Harwell, Central Laser Facility, Rutherford Appleton Laboratory Science , Technology Facilities Council , Harwell-Oxford, Didcot OX11 0FA , United Kingdom.
Institute of Applied Optics, Friedrich-Schiller-University Jena , Max-Wien Platz 4 , 07743 Jena , Germany.
Leibniz Institute of Photonic Technology e.V. , Albert-Einstein-Straße 9 , 07745 Jena , Germany.


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.


Diffusion; STED−FCS; lipids; plasma membrane; scanning FCS; simultaneous scanning

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