Rollins, G.C. et al. Proc. Natl. Acad. Sci. USA 112, E110–E118 (2015).

Super-resolution imaging techniques such as photoactivated localization microscopy (PALM) are becoming widely used in biology. However, determining protein-complex stoichiometry using PALM remains challenging, in part because of blinking associated with fluorescent proteins. Rollins et al. developed a stochastic approach to measure stoichiometry using PALM data, based on continuous-time aggregated Markov model techniques developed to study ion channels. This approach models fluorescent protein activation, blinking and photobleaching to determine the number of fluorophores in a complex. Using simulated and experimental data, the researchers benchmarked their method and demonstrated its utility in deriving the number of fluorophores in a complex and the error associated with that estimate. The method provides a promising new approach to solving the molecular counting problem in super-resolution microscopy.