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Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

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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.

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Authors and Affiliations

  1. Rudolf-Virchow-Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Street, Würzburg, Germany

    Ralf Steinmeyer, Andrey Noskov, Isabell Weber & Gregory S. Harms

  2. Institute of Pharmacology, University of Würzburg, Versbacher Street, Würzburg, Germany

    Cornelius Krasel & Christian Dees

Authors
  1. Ralf Steinmeyer
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  2. Andrey Noskov
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  3. Cornelius Krasel
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  4. Isabell Weber
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  5. Christian Dees
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  6. Gregory S. Harms
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Correspondence to Gregory S. Harms.

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Authors contributed equally to this article.

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Steinmeyer, R., Noskov, A., Krasel, C. et al. Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization. J Fluoresc 15, 707–721 (2005). https://doi.org/10.1007/s10895-005-2978-4

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  • DOI: https://doi.org/10.1007/s10895-005-2978-4

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