FLUORESCENCE CORRELATION SPECTROSCOPY AND PHOTOBLEACHING RECOVERY.

Fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) are closely related methods that can be used to measure rates of molecular transport and chemical reaction via observations of fluorescence changes in small open regions of a system. In FCS the fluorescence from the observation region is recorded over time while the sample rests in thermodynamic equilibrium. Spontaneous microscopic fluctuations of the numbers of fluorescent molecules in the observation region yield corresponding fluctuations in the measured fluorescence. The lifetimes of the fluctuations are determined in a statistical sense by the rates of molecular transport (diffusion, drift, or flow) into and out of the observation region and by the rates of chemical reactions among the system components. The effects of the photobleaching reaction on FPR results and in generating differences between FCS and FPR are a principal topic of this review.