On the Equivalence of Fcs and Frap: Simultaneous Lipid Membrane Measurements by Imaging Fcs

Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery after Photobleaching (FRAP) are probably the most widely used methods to determine diffusion coefficients in biological samples. However, it has already been noted in the past that the two methods do not necessarily provide the same results. This has typically been explained by the different spatial range over which the two methods measure diffusion. Diffusion at small distances, as measured by FCS, presumably measures local diffusion coefficients not hindered by any macromolecular structures. FRAP, on the other hand measures diffusion over long distances and thus of particles within larger molecular structures, e.g. the cytoskeleton, or diffusion hindered by the interaction/binding of the probe with other molecules. In addition, FRAP uses a large laser power during the bleach pulse, raising concerns about photodamage of the sample. And although, several studies in the past have compared FCS and FRAP, none of them have measured both methods simultaneously. A method to achieve simultaneous measurements on the same sample is Imaging Total Internal Reflection FCS (ITIR-FCS). ITIR-FCS is a multiplexed FCS method that measures correlation function in each pixel of an image with a field of view that can be as large as the area typically used for FRAP measurements. Here we use ITIR-FCS and FRAP in parallel on supported lipid bilayers and live cell membranes and compare the experiments to simulations (Fig. 1). The results show that FCS and FRAP obtain the same diffusion coefficients, the bleaching pulse has little or no influence on the molecular dynamics, but the exact FRAP results, unlike FCS, are very sensitive to the data evaluation procedure.