title: Improved fluorescent proteins for single-molecule research in molecular tracking and co-localization.
abstract: Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and >50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores.
authors.affiliation: Rudolf-Virchow-Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Street, Würzburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Steinmeyer
authors.forename: Andrey
authors.initials: A
authors.lastname: Noskov
authors.forename: Cornelius
authors.initials: C
authors.lastname: Krasel
authors.forename: Isabell
authors.initials: I
authors.lastname: Weber
authors.forename: Christian
authors.initials: C
authors.lastname: Dees
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Harms
mesh_terms: D000818:Animals; D001692:Biological Transport; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D038761:Photobleaching; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein