Molecular Counting with Localization Microscopy: A Bayesian Estimate Based on Fluorophore Statistics

Abstract Superresolved localization microscopy has the potential to serve as an accurate, single-cell technique for counting the abundance of intracellular molecules. However, the stochastic blinking of single fluorophores can introduce large uncertainties into the final count. Here we provide a theoretical foundation for applying superresolved localization microscopy to the problem of molecular counting based on the distribution of blinking events from a single fluorophore. We also show that by redundantly tagging single molecules with multiple, blinking fluorophores, the accuracy of the technique can be enhanced by harnessing the central limit theorem. The coefficient of variation then, for the number of molecules M estimated from a given number of blinks B , scales like ∼ 1 / N l , where N l is the mean number of labels on a target. As an example, we apply our theory to the challenging problem of quantifying the cell-to-cell variability of plasmid copy number in bacteria.