Funds are requested for a pulse laser light source and fast detector for time-resolved fluorescence spectroscopy. The source will be a mode-locked Neodinium-YAG laser, and a sycronously pumped dye laser cavity dumped to a frequency doubler. This source will provide 10 psec pulses frm 270 to 450 nm, with a repetition rate of 800 kHz. This source will be added to an existing single photon counting lifetime instrument shared by Professors Lakowicz, Bucci, Steiner and Inesi. The existing instrument, from Edinburgh Instruments, uses a flash lamp source, which limits the data acquisition rate to a maximum of 2000 cps (2% of the 40 kHz lamp rate). The pulse width of 1.5 nsec limits our ability to measure decays of fluorescence anisotropy. The low rate of data acquisition limits the statistical accuracy obtainable in reasonable data acquisition time, which hinders the resolution of distance distributions. The 40 kHz acquisiton rate (800 kHz lamp rate) provided by the laser source will substantially improve the signal-to-noise. The improved time response (less than 100 psec) will provide improved resolution of rapid anisotropy decays. High resolution of intensity and of anisotropy decays is essential for each of the principal users. Dr. Lakowicz is studying on the dynamics of tryptophan and tyrosine residues in proteins, pressure effects on proteins, and distance distributions by energy transfer. Dr. Steiner is examining the solution properties of calmodulin and troponin-C. Dr. Bucci is examining the dynamics of flavoproteins and hemoglobin. Dr. Inesi is examining kinetic models of ATPase based on the short-lived TNP-ATP probe, and the distance between sites on the ATPase. The existing dual monochromator instrument will serve admirably with the laser source. Software is available for time-resolved intensities, anisotropies, and emission spectra. We have Global Software from Professor L. Brand, and Moments Software from E. Small and I Isenberg. A procedure for time-sharing and payment of service contracts for five years has been agreed upon by the users. Dr. Lakowicz now has hands-on experience with a mode- locked cavity-dumped frequency-doubled system, and will perform the initial set up. The School of Medicine has agreed to supply $3000 per year for three years for maintenance. Experienced individuals are available six days per week to provide advice for users of the new instrument.
