Fluorescence Correlation Spectroscopy in living cells: a practical approach

Fluorescence correlation spectroscopy is a single-molecule fluorescence technique used to monitor molecular dynamics. When applied to living cells, FCS has been used to decipher the diffusion times of protein and lipid molecules, to determine the concentrations of a particular molecule at a specific cellular location, to determine kinetic parameters such as on/off rates constants and binding coefficients for protein/protein interactions [1]. The basic principle for FCS is that a fluorescent molecule emits photons while moving through a confocal volume illuminated by a laser (Figure 1). The number of photons that can be collected from the confocal volume depends on the diffusion time of the fluorescent molecules (which is a function of the mass of the molecule), the concentration of molecules, the quantum yield of the fluorophore attached to the molecule and finally the size of the illuminated volume, which is dependent on the instrument [2]. Typically a small diffraction limited confocal volume of 0.3μm × 1μm (∼size of an E. coli) is excited with a laser beam and then the emitted photons are collected and counted on a photodiode detector. These measurements are then used to calculate and display autocorrelation and cross correlation functions, coincidence diagrams, photon count histograms, count rate diagrams and pulse density histograms from which diffusion times, on/off rates etc can be extracted using data fitting and modeling software [3]. Figure 1 sketch of the optical setting for an FCS measurement. In this protocol, we present the basic design of an FCS measurement in living cells, and address critical issues in experiment design (from the choice of dyes or cells to data analysis). The user must be aware that FCS requires subsequent adjustments in the experimental design as well as the data analysis to address successfully specific biological questions. Comments after the basic protocol will present classic pitfalls and troubleshooting tips to help this.