Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

ABSTRACT: Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we de

SUBMITTER: Nieuwenhuizen RP 

PROVIDER: S-EPMC4439177 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature