Search Results
Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion
2018-7-20, Schneider, Falk, Waithe, Dominic, Lagerholm, B. Christoffer, Shrestha, Dilip, Sezgin, Erdinc, Eggeling, Christian, Fritzsche, Marco
Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.
Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer
2018-7-24, Urbančič, Iztok, Garvas, Maja, Kokot, Boštjan, Majaron, Hana, Umek, Polona, Cassidy, Hilary, Škarabot, Miha, Schneider, Falk, Galiani, Silvia, Arsov, Zoran, Koklic, Tilen, Matallanas, David, Čeh, Miran, Muševič, Igor, Eggeling, Christian, Štrancar, Janez
Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane’s disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.
Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging
2018-6-12, Schneider, Falk, Waithe, Dominic, Galiani, Silvia, Bernardino de la Serna, Jorge, Sezgin, Erdinc, Eggeling, Christian
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.