Single-pulse picosecond determination of 735 nm fluorescence risetime in spinach chloroplasts

Abstract

1. Introduction Within the last few years there have been extensive investigations of the fluorescence from photosynthetic systems using picosecond lasers as excitation sources [l-l 11. Such studies have established that the quenching of the prompt fluorescence, with increasing incident excitation intensity employing a single pulse, arises primarily from singlet--singlet exciton annihila- tion [2,3,7-91. When the mode of excitation consists of a train of pulses or of a microsecond laser pulse, there exists additional quenching of the fluorescence quantum yield arising from long-lived quenchers, presumably triplet excitons [2,3,10] , which either arise from intersystem crossing from the singlet mani- fold or from random recombination of electrons and holes formed from the autoionization final state channel of singlet fusion [8]. Recently, Geacintov et al. [8] have shown that the quantum yield quenching at emission wavelengths of 685 nm and 735 nm were identical, within experimental error, when single picosecond excitation pulses were employed. This identity in the quenching curves was interpreted in terms of the tripartite fluorescence model with bimolecular singlet fusion occurring exclusively within the light harvesting antenna system. Strong support for this interpretation was provided by the observation of an intensity-independent (for intensities below 10” photons cm-* per pulse) 316 lifetime for the 735 nm emission [l 1] . Presumably, singlet fusion reactions are either inoperative or inefficient within the PS I antenna chlorophyll molecules, at least for intensities below lo’* photons cm-’ per pulse. Within the light harvesting and PS II antenna pigments which give-rise to the 685 nm emission, singlet exciton fusions do give rise to a strong decrease of the fluorescence lifetime with increasing intensity. It was concluded that the pigments which are responsible for the 735 nm emission derive their energy by singlet exciton transfer from the light harvesting system and not by direct photon absorp- tion at 530 nm. In this paper we measure the risetime for the 735 nm emission and identify this time lag with the transfer rate from the light harvesting system to the PS I pigment molecules which give rise to the 735 nm fluorescence band at low temperatures. 2. Materials and methods The experimental arrangement for fluorescence lifetime measurements was similar to that reported previously [3] . A 1060 nm, 30 ps pulse was selected from the pulse train emitted by a modelocked Nd:YAG laser, frequency shifted to 530 nm by passage through a KDP crystal, and then was allowed to excite the spinach samples. The samples, chloroplasts prepared from spinach leaves as described in [ 121 , were