"uuid","repository link","title","author","contributor","publication year","abstract","subject topic","language","publication type","publisher","isbn","issn","patent","patent status","bibliographic note","access restriction","embargo date","faculty","department","research group","programme","project","coordinates" "uuid:dc2964e9-2dea-4499-afa2-aae2f968c683","http://resolver.tudelft.nl/uuid:dc2964e9-2dea-4499-afa2-aae2f968c683","Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry","Nieuwenhuizen, R.P.J.; Bates, M.; Szymborska, A.; Lidke, K.A.; Rieger, B.; Stallinga, S.","","2015","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 demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.","stoichiometry; bleaching; fluorescence imaging; photons; absorption spectroscopy; fluorescence microscopy; oligomers; glass; OA-Fund TU Delft","en","journal article","Public Library of Science","","","","","","","","Applied Sciences","ImPhys/Imaging Physics","","","",""