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Fluorescence Correlation Spectroscopy (FCS) is an important technique for understanding molecular dynamics and motion on timescales ranging from nanoseconds to seconds. The high concentrations found in some biological systems reduce the significance of FCS, as too many fluorophores are present within a standard confocal volume. Combining FCS with Stimulated Emission Depletion (STED) is one technique to overcome this problem by reducing the observation volume in the sample, reducing the number of molecules within this volume. This technique also allows the observation volume to be tuned to access additional information, such as the parameters which characterize hindered diffusion. In addition, by utilizing galvoscanners, scanning FCS can increase the number of transits recorded and reduce the residence time of each molecule, increasing the statistics and reducing the effects of photobleaching respectively. This technique is also useful for increasing the number of transits observed for slowly diffusing species at low concentrations, such as observed in membranes. Measuring multiple species simultaneously saves time and allows interactions between molecules to be investigated, which may not be clear from multiple experiments investigating single species. By exploiting the lifetime information available from a microscope equipped with Time Correlated Single Photon Counting (TCSPC) hardware, pattern matching can be used to separate similar fluorophores allowing up to three superresolved species to be resolved using a single STED laser. This pattern matching analysis can be combined with STED-FCS and scanning FCS to investigate complex diffusion in membranes. |
