Inferring subunit stoichiometry from single molecule photobleaching

Single molecule photobleaching is a powerful tool for determining the stoichiometry of protein complexes. By attaching fluorophores to proteins of interest, the number of associated subunits in a complex can be deduced by imaging single molecules and counting fluorophore photobleaching steps. Because some bleaching steps might be unobserved, the ensemble of steps will be binomially distributed. In this work, it is shown that inferring the true composition of a complex from such data is nontrivial because binomially distributed observations present an ill-posed inference problem. That is, a unique and optimal estimate of the relevant parameters cannot be extracted from the observations. Because of this, a method has not been firmly established to quantify confidence when using this technique. This paper presents a general inference model for interpreting such data and provides methods for accurately estimating parameter confidence. The formalization and methods presented here provide a rigorous analytical basis for this pervasive experimental tool.