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Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging
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 and interface effects during preparation of all-solid microstructured fibers
All-solid microstructured optical fibers (MOF) allow the realization of very flexible optical waveguide designs. They are prepared by stacking of doped silica rods or canes in complex arrangements. Typical dopants in silica matrices are germanium and phosphorus to increase the refractive index (RI), or boron and fluorine to decrease the RI. However, the direct interface contact of stacking elements often causes interrelated chemical reactions or evaporation during thermal processing. The obtained fiber structures after the final drawing step thus tend to deviate from the targeted structure risking degrading their favored optical functionality. Dopant profiles and design parameters(e.g., the RI homogeneity of the cladding) are controlled by the combination of diffusion and equilibrium conditions of evaporation reactions. We show simulation results of diffusion and thermal dissociation in germanium and fluorine doped silica rod arrangements according to the monitored geometrical disturbances in stretched canes or drawn fibers. The paper indicates geometrical limits of dopant structures in sub-μm-level depending on the dopant concentration and the thermal conditions during the drawing process. The presented results thus enable an optimized planning of the preform parameters avoiding unwanted alterations in dopant concentration profiles or in design parameters encountered during the drawing process.