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

Handle URI:
http://hdl.handle.net/10754/325368
Title:
A general approach to break the concentration barrier in single-molecule imaging
Authors:
Loveland, Anna B.; Habuchi, Satoshi ( 0000-0002-6663-2807 ) ; Walter, Johannes C.; van Oijen, Antoine M.
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 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. © 2012 Nature America, Inc. All rights reserved.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Loveland AB, Habuchi S, Walter JC, van Oijen AM (2012) A general approach to break the concentration barrier in single-molecule imaging. Nature Methods 9: 987-992. doi:10.1038/nmeth.2174.
Publisher:
Springer Nature
Journal:
Nature Methods
Issue Date:
9-Sep-2012
DOI:
10.1038/nmeth.2174
PubMed ID:
22961247
PubMed Central ID:
PMC3610324
Type:
Article
ISSN:
15487091
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorLoveland, Anna B.en
dc.contributor.authorHabuchi, Satoshien
dc.contributor.authorWalter, Johannes C.en
dc.contributor.authorvan Oijen, Antoine M.en
dc.date.accessioned2014-08-27T09:49:27Zen
dc.date.available2014-08-27T09:49:27Zen
dc.date.issued2012-09-09en
dc.identifier.citationLoveland AB, Habuchi S, Walter JC, van Oijen AM (2012) A general approach to break the concentration barrier in single-molecule imaging. Nature Methods 9: 987-992. doi:10.1038/nmeth.2174.en
dc.identifier.issn15487091en
dc.identifier.pmid22961247en
dc.identifier.doi10.1038/nmeth.2174en
dc.identifier.urihttp://hdl.handle.net/10754/325368en
dc.description.abstractSingle-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 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. © 2012 Nature America, Inc. All rights reserved.en
dc.language.isoenen
dc.publisherSpringer Natureen
dc.rightsUsers may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsen
dc.rightsArchived with thanks to Nature Methodsen
dc.subjectdeoxyribonuclease Ien
dc.subjectXenopus proteinen
dc.subjectconcentration (parameters)en
dc.subjectdiffusionen
dc.subjectDNA replicationen
dc.subjectmolecular imagingen
dc.subjectphotoactivationen
dc.subjectprotein analysisen
dc.subjectprotein bindingen
dc.subjectprotein immobilizationen
dc.subjectXenopus laevisen
dc.subjectDiffusionen
dc.subjectDNA Replicationen
dc.subjectFlap Endonucleasesen
dc.subjectLuminescent Proteinsen
dc.subjectMicroscopy, Fluorescenceen
dc.subjectProliferating Cell Nuclear Antigenen
dc.subjectEukaryotaen
dc.subjectXenopus laevisen
dc.titleA general approach to break the concentration barrier in single-molecule imagingen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalNature Methodsen
dc.identifier.pmcidPMC3610324en
dc.eprint.versionPost-printen
dc.contributor.institutionDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United Statesen
dc.contributor.institutionGraduate Program in Biophysics, Harvard University, Cambridge, MA, United Statesen
dc.contributor.institutionDepartment of Biochemistry, Brandeis University, Waltham, MA, United Statesen
dc.contributor.institutionZernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlandsen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorHabuchi, Satoshien

Related articles on PubMed

All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.