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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.
Background Reduction in STED-FCS Using a Bivortex Phase Mask
Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale. Copyright © 2020 American Chemical Society.
Adaptive optics allows STED-FCS measurements in the cytoplasm of living cells
Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (z) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.