Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging
| Autor: | Schneider, F, Waithe, D, Galiani, S, De la Serna, JB, Sezgin, E, Eggeling, C |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
DYNAMICS
Diagnostic Imaging Technology LATERAL DIFFUSION Chemistry Multidisciplinary Materials Science Lipid Bilayers Materials Science Multidisciplinary CONFINEMENT plasma membrane Physics Applied GPI-ANCHORED PROTEINS Diffusion lipids CELL-MEMBRANE ORGANIZATION STED−FCS MD Multidisciplinary Humans Nanoscience & Nanotechnology Science & Technology Microscopy Confocal STED-FCS Chemistry Physical Physics simultaneous scanning Cell Membrane FLUORESCENCE CORRELATION SPECTROSCOPY Chemistry LIPID RAFTS Nanomedicine Spectrometry Fluorescence Physics Condensed Matter PAIR CORRELATION Microscopy Fluorescence Physical Sciences PLASMA-MEMBRANE Science & Technology - Other Topics Nanoparticles lipids (amino acids peptides and proteins) scanning FCS |
| DOI: | 10.34657/9974 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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