Photobleaching, Mobility, and Compartmentalisation: Inferences in Fluorescence Correlation Spectroscopy

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter pB = σ P/D (where σB is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when pB ≪ 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like σBP/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.