9319032 Lakowicz Stationary and time-resolved fluorescence spectroscopy are widely used in Biochemistry and Biophysics. The information content of the data is determined by the many lifetime of the excited state, which determines the time available for dynamic processes to affect the emission spectral parameters. We propose to evaluate the use of "light quenching" upon illumination of the samples with long-wavelength non-absorbed light, to alter the decay times and photoselected orientation of the excited state population. In this exploratory project we will evaluate the prospects for light quenching using modern laser sources, as applied to steady-state, time-domain and frequency-domain measurements. We propose the following goals: 1. Determine the extent of light quenching obtainable with fluorophores commonly present in proteins, membranes and nucleic acids. 2. Evaluate the use of light quenching and steady-state fluorescence measurements based on the principle of "lifetime-resolved" emission spectra and anisotropy decays. 3. Evaluate the use of light quenching to modify the orientation of the photoselected population, with emphasis on optical geometries which can generate oriented (non- symmetric) excited state populations. 4. Develop the appropriate theoretical expressions and software for light quenching for one - and two - beam populations. 5. Examine light quenching of sensing probes, such as those used for Ca2+, pH and CI-, to determine the prospect for lifetime-resolved imaging in fluorescence microscopy, via light quenching. Light quenching offers a non-perturbing means of controlling the excited state population and orientation without the addition of chemical quenchers. Also, light quenching is rapidly reversible. This phenomena may have many unforeseen future applications, not only in steady- state and time-resolved fluorescence spectroscopy, but also in fluorescence microscopy and imaging.
