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Subnanosecond single photon counting fluorescence spectroscopy using synchronously pumped tunable dye laser excitation.

A synchronously pumped tunable dye laser has been constructed and interfaced with a modified Ortec 9200 photon counting system for the purpose of measuring subnanosecond relaxation phenomena. The dye laser excitation pulse, which has an intrinsic 35-ps FWHM for Rhodamine 6G, is 350 ps when measured by time-correlated single photon counting. This value appears to be characteristic of the transit time jitter in the RCA 8850 photomultiplier tube. Subnanosecond fluorescence lifetimes of Rhodamine B with KI as a quencher have been determined by deconvolution of photons counted versus elapsed time using the method of moments; the shortest lifetime measured was 68 ps. Various technical aspects of the system are discussed with emphasis on applications to biophysical problems.