Fluorescence anisotropy decays with minimal instrumental artifacts

摘要

We have constructed a fluorescence decay instrument with computer-automated optical components and data acquisition. Stepping motors control polarizer orientations, filter holders, retarder for incident intensity control, and four position sample changer. The changer uses a mechanical indexer for rapid, precise positioning, and has thermoelectric temperature control. Nitrogen flush and magnetic stirring are provided for all four cuvette positions. An automatic shutter protects the photomultiplier tube, closing automatically when the instrument is opened or the measured photon flux exceeds a predetermined limit. Optical sensors relay position information to the computer for all moving components. The instrument is controlled by a Windows-based program designed to accommodate users of widely varying ability. An inexperienced student can automatically run a complex anisotropy decay experiment with careful sensitivity corrections. Using simple editing functions, a more experienced user, on the other hand, can vary an experiment in minute detail. Automatic algorithms are used to home the instrument at the beginning of an experiment, to increase incident laser intensity until a specified count rate is achieved, and to maintain the count rate during a measurement. We also summarize here some instrumental artifacts common to time-resolved fluorescence data as well as approaches we have used to minimize their effects on recovered decay parameters.