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A general approach to break the concentration barrier in single-molecule imaging

Journal Article


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Abstract


  • Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion, and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA-replication protein Flap endonuclease 1 (Fen1) with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

Authors


  •   Loveland, Anna B. (external author)
  •   Habuchi, Satoshi (external author)
  •   Walter, Johannes C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2012

Citation


  • Loveland, A. B., Habuchi, S., Walter, J. C. & van Oijen, A. M. (2012). A general approach to break the concentration barrier in single-molecule imaging. Nature Methods, 9 (10), 987-992.

Scopus Eid


  • 2-s2.0-84867081368

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3183&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2165

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 987

End Page


  • 992

Volume


  • 9

Issue


  • 10

Place Of Publication


  • United Kingdom

Abstract


  • Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion, and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA-replication protein Flap endonuclease 1 (Fen1) with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

Authors


  •   Loveland, Anna B. (external author)
  •   Habuchi, Satoshi (external author)
  •   Walter, Johannes C. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2012

Citation


  • Loveland, A. B., Habuchi, S., Walter, J. C. & van Oijen, A. M. (2012). A general approach to break the concentration barrier in single-molecule imaging. Nature Methods, 9 (10), 987-992.

Scopus Eid


  • 2-s2.0-84867081368

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3183&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2165

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 987

End Page


  • 992

Volume


  • 9

Issue


  • 10

Place Of Publication


  • United Kingdom