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The measurement of fast fluorescence waveforms with a traditional sequential sampling technique results in an inaccurate determination of the fluorescence decay time(s) in the presence of fluorescence fading. This paper describes a random sampling technique that overcomes this difficulty. A suitable software for random sampling and averaging has been developed for a subnanosecond pulsed‐laser microfluorometer. Details of the random sampling routine are presented. The advantages of the random sampling technique as opposed to sequential sampling are discussed mathematically, and results of computer simulation for the two methods are shown.

A computer‐controlled system for time‐resolved fluorescence spectroscopy is described. The system provides the simultaneous acquisition of the fluorescence decay curve, of the time‐integrated emission spectrum, and of two time‐gated spectra. Gating within the fluorescence decay is provided by setting up windows in the output of the time‐to‐amplitude converter in single photon timing instrumentation. The gate parameters (position and width) are fully programmable. Depending on the laser source used, time resolutions between 45 and 150 ps are obtained. Therefore, in most situations no deconvolution procedures are required to elaborate the spectra. The system has been characterized with the aid of suitable test solutions. The results indicate that a correct choice of the gate parameters allows complementary information (in terms of both decay times and emission spectra) of the fluorescence parameters of molecular species in molecular systems.

The tetracycline dyes, particularly chlorotetracycline, have been employed as probes of membrane-associated calcium during the excitation process of nerve. Both squid giant axons, stained internally, and lobster nerves, stained externally, show a small increase in fluorescent light during the action potential. Increasing the calcium concentration bathing a lobster nerve leads to a larger optical signal. Adding fluoride ion to the inside of a squid axon, which might be expected to influence the internal calcium-ion concentration, also leads to a larger optical signal. Squid axons have been studied under conditions of voltage clamp and the hyperpolarizing response. Model studies were done with erythrocyte ghosts to clarify the influence of membranes and calcium on the fluores-cence of the tetracyclines. Chlorotetracycline may be monitoring calcium concentration associated with the inner surface of the nerve membrane.