Model-independent counting of molecules in single-molecule localization microscopy

Most biomolecular processes rely on tightly controlled stoichiometries, from the formation of molecular assemblies to cellular signaling. Single-molecule localization micro­scopy studies of fluorophore blinking offer a promising route to probe oligomeric states. Here we show that the distribution of the number of blinking events assumes a universal functional form, independent of photophysics, under relatively mild assumptions. The number of photophysical states, the kinetics of interconversion, and the fraction of active fluorophores enter as two or three constants. This essentially model-independent formulation allows us to determine molecule counts from fluorophore blinking statistics. The formulas hold even if the fluorophores have many different yet unresolved dark states, as long as there is only a single fluorescent state, or if there are different yet unresolvable fluorescent states, as long as there is only a single dark state. We demonstrate the practical applicability of this approach by quantifying the oligomerization states of membrane proteins tagged with the mEos2 fluorescent protein. We find that the model parameters, obtained by likelihood maximization, are transferable. With the counting statistics being independent of the detailed photophysics and its parameters being transferable, the method should be robust and broadly applicable to counting colocalized molecules in vivo and in vitro.