title: Simple calibration of TIR field depth using the supercoiling response of DNA.
abstract: The combination of single-molecule fluorescence and nanomanipulation techniques into a single experimental platform enables one to carry out correlative analysis of the composition and the activity of complex, multicomponent molecular systems. Here we describe implementation and calibration of such a combined system allowing simultaneous single-molecule force spectroscopy and fluorescence imaging of proteins acting on the DNA using magnetic trapping coupled with fluorescence excitation based on a Total Internal Reflection (TIR), or evanescent, field. We propose a simple and robust in situ method for calibration of the TIR field depth against the mechanical properties of nanomanipulated DNA, and which is made possible by the fact that the magnetic bead used to trap and nanomanipulate DNA and measure its conformation also exhibits autofluorescence in the TIR field. Indeed, the fact that the bead size is on the 1-micron scale does not preclude sensitive probing of an intensity field which decays exponentially on the 0.1micron-scale. We demonstrate the usefulness of this approach by mapping out TIR field depth as a function of the angle of incidence of the illuminating laser at the glass-water interface and showing that one recovers the expected theoretical relationship between field depth and angle of incidence.
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Duboc
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Evan T
authors.initials: ET
authors.lastname: Graves
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France; Ecole Normale Supérieure, Institut de Biologie de l'ENS (iBENS), INSERM, CNRS, PSL Research University, Paris, France. Electronic address: strick.terence@ijm.univ-paris-diderot.fr.
authors.forename: Terence R
authors.initials: TR
authors.lastname: Strick
mesh_terms: D002138:Calibration; D004247:DNA / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D000072760:Single Molecule Imaging / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D004247:DNA
keywords: Correlative methods; DNA; NanoCOSM; Single-molecule fluorescence; Single-molecule nanomanipulation; Single-molecules