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Molecule Counts in Localization Microscopy with Organic Fluorophores
Author(s) -
Karathanasis Christos,
Fricke Franziska,
Hummer Gerhard,
Heilemann Mike
Publication year - 2017
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201601425
Subject(s) - alexa fluor , fluorophore , microscopy , molecule , fluorescence , chemistry , resolution (logic) , fluorescence microscope , materials science , optics , physics , organic chemistry , artificial intelligence , computer science
Single‐molecule localization microscopy (SMLM) can be used to count fluorescently labeled molecules even when they are not individually resolved. We demonstrate SMLM molecule counting for nucleic acids labeled with the organic fluorophore Alexa Fluor 647 and imaged under photoswitching conditions. From the observed distributions of the number of fluorophore blinking events, we extract the number of fluorophores per spot using a statistical model. We validate the molecule counting method for single Alexa Fluor 647 fluorophores, and for trimers of Alexa Fluor 647 constructed on a DNA origami structure. This simple counting strategy enables quantitative super‐resolution imaging with organic fluorophores.