Absolute quantification of protein number and dynamics in single cells

Quantitative characterization of protein abundance and interactions in live cells is necessary to understand and predict cellular behavior. The accurate determination of copy number for individual proteins and heterologous complexes in individual cells is critical because small changes in protein dosage, often less than two-fold, can have strong phenotypic consequences. Here, we review the merits and pitfalls of different quantitative fluorescence imaging methods for single-cell determination of protein abundance, localization, interactions, and dynamics. In particular, we discuss how scanning number and brightness (sN&B) and its variation, Raster scanning image correlation spectroscopy (RICS), exploit stochastic noise in small measurement volumes to quantify protein abundance, stoichiometry, and dynamics with high accuracy.