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We have previously reported measurements of fluorescence lifetimes for both photosynthetic systems and chlorophyll solutions using a streak camera technique (1, 2) and picosecond excitation. Our lifetime measurements on antenna systems, however, as well as earlier results obtained using a picosecond resolution optical gate (3-5), indicate somewhat shorter decay times than measured with previous techniques (6-10). Our present experimental investigations of a possible intensity-dependent effect as the cause of these differences are prompted by the recent results of Mauzerall (11). He has shown that the quantum efficiency for the fluorescence emission from Chlorella decreases at higher pumping intensities for 7-ns duration excitation pulses, and he has interpreted the decrease in terms of a multitrap model of the photosynthetic unit and exciton-exciton collisions. In order to assess the respective roles that singlet and triplet excitons play in the kinetics of these interactions, it is desirable to repeat these measurements using a much shorter excitation pulse and under conditions comparable to the previously mentioned picosecond experiments. In this letter we report results for the quantum efficiency of Chlorella as a function of intensity, but with picosecond excitation. A single 20 ps pulse has been selected from a mode-locked laser pulse train for these measurements. Results show a drop of quantum efficiency with intensity in agreement with results of Mauzerall. Previous picosecond fluorescence lifetime measurements must be reinterpreted in view of this nonlinear optical effect.In order to investigate the possibility that the somewhat shorter lifetimes obtained by picosecond techniques for antenna systems originate from nonlinear optical effects present at high-energy densities, a different experimental arrangement than we used previously for lifetime measurements is required for quantum efficiency measurements. Previously, all the pulses in a mode-locked pulse train were allowed to excite the sample, and the fluorescence produced by one of the pulses could be examined. When making intensity-dependent quantum efficiency measurements, such a pulse train technique may lead to difficulties in interpretation. For example, pulses exciting the sample prior to the pulse chosen for investigation may populate the reaction centers, leave a residual population of antenna chlorophyll molecules in the triplet state, or otherwise change the system. Thereafter, singlets generated by the pulse to be exam-BIOPHYSICAL JOURNAL VOLUME 16 1976 93

New data on intensity-dependent lifetimes indicate that all previous in vivo fluorescence studies of chlorophyll by picosecond techniquies must be reinterpreted. Anomalously short lifetimes result from high-intensity effects due to exciton-excition annihilation processes. Measurements in Chlorella pyrenoidosa with single-pulse, low-intensity excitation indicate a longer "true" lifetime of 650 +/- 150 picoseconds.

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