A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatus

Abstract

A new spectrophotometric technique has been developed in which the absorption is sampled by short flashes produced by an optical parametric oscillator (OPO) pumped by the third harmonic of a Nd:YAG laser. Continuously tunable monochromatic flashes (6 ns duration, 1 nm bandwidth) are obtained in a domain ranging from 415 to 680 nm and 730 to 2000 nm. Light-induced absorption changes can be analyzed in a time window ranging from 10 ns to 100 s, with a 10−5 noise level. This new technique opens areas of research which are not accessible to previously existing spectrophotometers. Indeed, the high energy of the detecting flashes allows the analysis of samples of large optical density such as leaves, in the blue region of the spectrum. A few examples of application of this method to the analysis of electron transfer reactions in different types of photosynthetic apparatus (leaves, unicellular algae, and bacteria) are presented. This technique is also well suited to analyze the photodissociation of ligands associated with hemoproteins, in solution or in living cells.