Nanoscopic Stoichiometry and Single‐Molecule Counting

Single‐molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here, a method for molecular counting is presented with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. A guide to performing this newly developed technique is provided, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLM's utility for optical “‐omics” analysis. Single‐molecule localization microscopy could revolutionize proteomic and genomic analyses by providing information on the abundance and stoichiometry of proteins or nucleic acids interacting at spatial scales below the diffraction limit of light. Here, a new technique for molecular counting with organic dyes is described, many of the challenges and pitfalls are highlighted, and the method is benchmarked on DNA origami.