Single Molecule Detection of Fluorescent Probes

点击翻译按钮获取中文摘要

This research will focus on the development of a novel and ultrasensitive apparatus for the detection of fluorescent probes. A detection system will be constructed that is sensitive enough to observe the fluorescence emitted by individual fluorophores. The new concept of single molecule counting or gating will be used to provide the necessary discrimination against Raleigh, Raman and detector background. To develop this detection system and to demonstrate the detection of individual fluorescent molecules in solution, the laser excited fluorescence from fluorophores in a flowing sample system will be studied. In the second part of this project, two-dimensional laser scanner systems will be constructed, utilizing this detection system, which will obtain images of the location of individual fluorophores on substrates such as slides, gels and films. The concepts of auto and cross correlation of multiple images will be used to provide further discrimination against spatially random background. Initial experiments on individual fluorophores and on fluorescent labeled antibodies or fluorescent beads in thin films will be used to demonstrate the imaging technique. Feasibility experiments exploring the sensitivity of this new technique for finding labeled protein or DNA bands on gels or fluorescent antibody conjugates with bacteria will be performed. The improved analytical sensitivity that will be provided by this methodology will be particularly valuable because it will increase the speed and sensitivity with which immunofluorescence assays and fluorescence-detected DNA sequencing gels can be performed. Significant future applications may include immunofluorescence detection of individual cell surface receptors or markers and the location of DNA probes on chromosomes. The detection of individual cell receptors or markers by fluorescent labeling will enable investigators of cellular and molecular processes to understand these processes more accurately. The complex molecular makeup of cells leads to difficulties identifying a specific molecule separately from a background of many different molecules. Detection improvement will benefit many areas of biology such as cell physiology, neurobiology, and biophysical cytology.