Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview

Chlorophyll (Chi) a fluorescence originates in close vicinity to the sites where light energy is transformed into chemically fixed energy. The same excitation states that give rise to fluorescence emission also participate in photochemical energy conversion. These features render Chi fluorescence aunique indicator of photosynthesis. During the past 15 years there has been remarkable progress in Chi fluorescence research. In practical applications, Pulse-Amplitude-Modulation (PAM) fluorometry in conjunction with the saturation pulse method has been particularly successful. This chapter outlines the principles of PAM fluorometry and saturation pulse method. Some examples of typical applications are given. Also the limits of the method are outlined, with emphasis on the fact that absolute assessment of photosynthetic parameters is complicated by various factors, while relative changes can be assessed with high reliability. Particular attention is given to the theoretical foundation of the method, at a level, which can be also understood by non-specialists and students. A pivotal role in the determination of Photosystem (PS) II quantum yield by fluorescence measurements is played by the maximal fluorescence yield, Fm, proper measurement of which has been a matter of controversy. This is related to a large intrinsic heterogeneity of variable Chi fluorescence, which is revealed in the polyphasic rise of fluorescence yield upon the onset of saturating light. Arguments are put forward in favor of Fm determination after full reduction of the plastoquinone pool, including the secondary quinone acceptor of PS II, Q_B, the oxidized form of which can support in my view a particular type of nonphotochemical quenching, when the primary quinone acceptor, Q_A, is reduced. Possible mechanisms and consequences of this ‘Q_B-quenching’ are discussed. Some PAM fiuorometers for special applications are described, including systems for phytoplankton analysis, for investigations at the single cell level (epifluorescence microscopy and microfiber technique) and for imaging of photosynthetic activity.