Quantitative Fluorescence Correlation Spectroscopy in 3D Systems under Stimulated Emission Depletion Conditions

Super-resolution fluorescence microscopy is becoming a widely available, standard tool in biophysical research. The leading deterministic approach, stimulated emission depletion (STED), can enhance the capabilities of various fluorescence techniques, including fluorescence correlation spectroscopy (FCS). Until now, STED-FCS has been successfully applied to diffusion studies in 2D systems such as membranes. Severe deficiencies, including overestimation of the detected number of probes as well as underestimation of their diffusion coefficients (both parameters differing from the expected values by up to an order of magnitude) impeded STED-FCS studies in solutions. Here, we introduce a realistic 3D model of the detection volume for STED-FCS and use it to resolve the apparent inconsistencies. We validate the model experimentally, recovering the number and diffusion rates of freely diffusing probes. We define the limitations of the method and provide simple guidelines for experiment design and data analysis. The proposed approach should prove useful for particle mobility and reaction kinetics studies in polymer solutions as well as bulk biomimetic and biological systems, especially when reactant concentrations exceeding 100 nM are required.