title: Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.
abstract: Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Haocheng
authors.initials: H
authors.lastname: Zheng
authors.forename: Lori S
authors.initials: LS
authors.lastname: Goldner
authors.forename: Sanford H
authors.initials: SH
authors.lastname: Leuba
mesh_terms: D016477:Artifacts; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D045566:DNA, Cruciform / Q000737:chemistry*; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007834:Lasers; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D009707:Nucleosomes / Q000737:chemistry*; D012984:Software
chemical_list: D045566:DNA, Cruciform; D009707:Nucleosomes