title: Single-molecule localization microscopy using mCherry.
abstract: We demonstrate the potential of the commonly used red fluorescent protein mCherry for single-molecule super-resolution imaging. mCherry can be driven into a light-induced dark state in the presence of a thiol from which it can recover spontaneously or by irradiation with near UV light. We show imaging of subcellular protein structures such as microtubules and the nuclear pore complex with a resolution below 40 nm. We were able to image the C-terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling. The photon yield for mCherry is comparable to that of the latest optical highlighter fluorescent proteins. Our findings show that the widely used mCherry red fluorescent protein and the vast number of existing mCherry fusion proteins are readily amenable to super-resolution imaging. This obviates the need for generating novel protein fusions that may compromise function or the need for external fluorescent labeling.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL (UK); Institute of Biochemistry, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich (Switzerland). christian.winterflood@kcl.ac.uk.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Winterflood
authors.forename: Helge
authors.initials: H
authors.lastname: Ewers
mesh_terms: D002460:Cell Line; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D000097573:Red Fluorescent Protein; C094157:POM121 protein, human
keywords: fluorescent labeling; mCherry; nuclear pore; protein structures; super-resolution imaging