A laser light source is requested to upgrade a flash-lamp nanosecond fluorometer now available in the Biology department. The main benefit to be accrued by addition of this mode-locked laser is its high repetition rate. Fluorescence decay curves that take two hours to accumulate at present would be obtainable in about two minutes. In addition, the tunable dye laser will provide a wider range of wavelengths. The laser light source is more intense than a flash lamp, allowing the introduction of a microscope into the optical system. The timing profile of the laser is in the picosecond time range so that the effective pulse width is not limited by the light source. Several ongoing projects would benefit from the use of the upgraded pulse fluorometer and numerous experiments that are not possible at present would become feasible. The ability to obtain a large number of decay curves during a reasonable time span is crucial. The simultaneous analysis of up to several hundred related decay curves (global analysis) allows the investigation of microheterogeneity in proteins and membranes. Mechanisms of excited-state reactions may be discerned that would be unapproachable from the analysis of only a few individual decay curves. Systems to be investigated include protein interactions and conformational changes of alcohol dehydrogenase, proteins of the PTS system, S. nuclease, t-RNA and the uvr protein systems. Additional areas to be investigated with the aid of this instrument include the fluidity of bilayers and lipid domains, cell surfaces and receptors and the transfer of lipids from liposomes to cells. The mechanisms involved in the fluorescence of potential sensitive probes will be studied in model and neurobiological systems. The instrument will be supported by modest user fees, if necessary.
