Overview of Fluorescence Protocols: Theory, Basic Concepts, and Practice

A newly developed fluorescence measuring system is employed for the recording of chlorophyll fluorescence induction kinetics (Kautsky-effect) and for the continuous determination of the photochemical and non-photochemical components of fluorescence quenching. The measuring system, which is based on a pulse modulation principle, selectively monitors the fluorescence yield of a weak measuring beam and is not affected even by extremely high intensities of actinic light. By repetitive application of short light pulses of saturating intensity, the fluorescence yield at complete suppression of photochemical quenching is repetitively recorded, allowing the determination of continuous plots of photochemical quenching and non-photochemical quenching. Such plots are compared with the time courses of variable fluorescence at different intensities of actinic illumination. The differences between the observed kinetics are discussed. It is shown that the modulation fluorometer, in combination with the application of saturating light pulses, provides essential information beyond that obtained with conventional chlorophyll fluorometers. Show more

A number of useful photosynthetic parameters are commonly derived from saturation pulse-induced fluorescence analysis. We show, that q(P), an estimate of the fraction of open centers, is based on a pure 'puddle' antenna model, where each Photosystem (PS) II center possesses its own independent antenna system. This parameter is incompatible with more realistic models of the photosynthetic unit, where reaction centers are connected by shared antenna, that is, the so-called 'lake' or 'connected units' models. We thus introduce a new parameter, q(L), based on a Stern-Volmer approach using a lake model, which estimates the fraction of open PS II centers. We suggest that q(L) should be a useful parameter for terrestrial plants consistent with a high connectivity of PS II units, whereas some marine species with distinct antenna architecture, may require the use of more complex parameters based on intermediate models of the photosynthetic unit. Another useful parameter calculated from fluorescence analysis is Phi(II), the yield of PS II. In contrast to q(L), we show that the Phi(II) parameter can be derived from either a pure 'lake' or pure 'puddle' model, and is thus likely to be a robust parameter. The energy absorbed by PS II is divided between the fraction used in photochemistry, Phi(II), and that lost non-photochemically. We introduce two additional parameters that can be used to estimate the flux of excitation energy into competing non-photochemical pathways, the yield induced by downregulatory processes, Phi(NPQ), and the yield for other energy losses, Phi(NO). Show more