Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Abstract

MotivationMany recent advancements in single molecule localisation microscopy exploit the stochastic photo-switching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. ResultsModelling the photo-switching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localisations from a single photo-switching fluorophore. This is then extended to provide the probability distribution for the number of localisations in a dSTORM experiment involving an arbitrary number of molecules. We demonstrate that when training data is available to estimate photo-switching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localisations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein Linker for Activation of T cells (LAT) on the cell surface of the T cell immunological synapse. AvailabilitySoftware available at https://github.com/lp1611/mol_count_dstorm.