Using fluorescence fluctuation spectroscopy to quantify molecular dynamics within cells

Originally developed for the field of physical chemistry, fluorescence fluctuation spectroscopy (FFS) has evolved into a family of methods to quantify concentrations, diffusion rates and interactions of fluorescently labeled molecules. Combining microscopy with the ability to measure molecules at the nanomolar level allows for the study of biological processes with high sensitivity even within the living cell. In this protocol, the principles of how to set up these types of experiments are described including calibration samples, challenges and specific cellular applications. The predecessor to FFS, fluorescence correlation spectroscopy (FCS) will be discussed which measures diffusion and concentrations at a user‐defined point within the cell. Furthermore, an imaging variant, number and brightness analysis (N&B), will be shown that can be used to quantify molecular oligomerization. Overall, this protocol provides a foundation for using FFS and associated techniques to examine molecular dynamics within cells.