Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions |
| |
Authors: | Zheng Haocheng Goldner Lori S Leuba Sanford H |
| |
Institution: | Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. |
| |
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. |
| |
Keywords: | |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|