title: Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate.
abstract: Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.
authors.forename: Nela
authors.initials: N
authors.lastname: Durisic
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.
authors.forename: Lara
authors.initials: L
authors.lastname: Laparra-Cuervo
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Sandoval-Álvarez
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.
authors.forename: Joseph Steven
authors.initials: JS
authors.lastname: Borbely
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.
authors.forename: Melike
authors.initials: M
authors.lastname: Lakadamyali
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis*; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D038761:Photobleaching / Q000528:radiation effects; D018009:Receptors, Glycine / Q000378:metabolism*; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018009:Receptors, Glycine; D049452:Green Fluorescent Proteins