Estimating the dynamic range of quantitative single-molecule localization microscopy.

In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here, we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on direct stochastic optical reconstruction microscopy (dSTORM) data sets of photoswitching Alexa Fluor 647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.