Fluorescence Techniques

Abstract

Fluorescence measurements have been used extensively in phycological research for estimating chlorophyll concentration in situ using techniques developed by Lorenzen (1966). Despite the fact that fluorescence from chlorophyll a in vivo is extremely low (≈ 3%, Latimer, Bannister & Rabinowitch, 1956) it provides a measure of pigment content with a sensitivity considerably greater than that of the limiting sensitivity of absorption measurements. Fluorescence measurements also provide information about competing mechanisms involved in the decay of excitation energy during photochemical reactions and, in recent years, have been used as an adjunct to gas exchange determinations, providing additional information on mechanisms underlying variations in carbon assimilation. Fluorescence techniques have an advantage over many conventional procedures in that they can provide a sensitive, nonintrusive probe of the photosynthetic apparatus. Considerable evidence now exists to indicate that alterations in fluorescence kinetics are closely correlated with changes in carbon metabolism and our present understanding of functional aspects of the photosynthetic apparatus have been largely brought about through the increased application of fluorometric techniques to photosynthesizing cells. For this reason, this chapter considers those applications of fluorescence that are relevant to an understanding of gas exchange responses. It is often customary to compare fluorescence and photosynthesis measurements made over a time scale of minutes and to ignore fast (micro-to millisecond) fluorescence kinetics. However, this approach neglects the fact that any one of a number of reactions varying on time scales of fractions of a second to minutes could have important effects on the rate of net CO₂ fixation. For this reason, we have included information on rapid fluorescence transients. Because the interpretation of fluorescence emission characteristics depends on a knowledge of the molecular processes underlying light emission, these are also discussed briefly. Whether fluorescence measurements in themselves are sufficient to characterize photosynthetic responses is currently uncertain, and fluorescence measurements are probably best used in combination with gas exchange determinations. Recent reviews and information on the theory and measurement of fluorescence parameters can be found in Papageorgiou (1975), Bolhar-Nordenkampf et al. (1989), Geacintov and Breton (1987), and in the book Light Emission by Plants and Bacteria edited by Govindjee et al. (1986). For details of current instrumentation, the reader should consult Mauzerall (1980), Schreiber (1983), Schreiber and Schliwa (1987), Ogren and Baker (1985), Schreiber, et al. (1989), and Bolhar-Nordenkampf et al. (1989). Details of the application of fluorescence measurements to ecological research are given in Schreiber and Bilger (1987), Bolhar-Nordenkampf et al. (1989), Krause and Somersalo (1989), Schafer and Björkman (1989) and Kiefer et al. (1989). Unfortunately, most of the more recent studies of fluorescence have been made on terrestrial vascular plants and further information on algae is required.