Introduction
Chl a fluorometry has long been recognized as a valuable, non-invasive technique for probing oxygenic photosynthesis. Over the past 15 years or so, increasingly capable Chl a fluorescence imaging systems have been developed by a number of research groups, for use at low resolution (Omasa et al., 1987; Daley et al., 1989; Fenton and Crofts, 1990; Genty and Meyer, 1995; Siebke and Weis, 1995; Scholes and Rolfe, 1996; Nedbal et al., 2000; Zangerl et al., 2002) and at the microscopic level (Oxborough and Baker, 1997a; Osmond et al., 1999; Küpper et al., 2000; Rolfe and Scholes, 2002). In addition, commercial Chl a fluorescence imaging systems have been developed by PSI (Brno, Czech Repuplic), Walz Systems (Effeltrich, Germany), and Technologica Ltd. (Colchester, UK).
The application of Chl a fluorescence imaging can be divided into two general areas; the study of heterogeneous phenomena and the screening of large numbers of samples. A number of publications describe the application of low resolution imaging systems to the study of heterogeneous patterns of photosynthetic performance in leaves. For example, during the onset of photosynthesis after a prolonged dark period (Bro et al., 1996), after fungal infection (Scholes and Rolfe, 1996), and during a sink–source transition (Meng et al., 2001). Examples of high resolution imaging include measurements from leaves during the onset of photosynthesis after a prolonged dark period (Oxborough and Baker, 1997b), after exposure to ozone (Leipner et al., 2001), and from heterogeneous populations of algal cells within intact biofilms (Oxborough et al., 2000). There is also one example of confocal microscopy being used to follow changes in fluorescence yield from grana and stroma lamellae (Osmond et al., 1999). Fluorescence imaging has also been used in the screening of algal mutant colonies with altered thylakoid electrochemical gradient (Bennoun and Beal, 1997), screening for non-photochemical mutants of Chlamydomonas sp. (Niyogi et al., 1997) and Arabidopsis sp. (Niyogi et al., 1998) and the detection of herbicide effects on the maximum efficiency of PSII photochemistry in Arabidopsis sp. and Agrostis tenuis, several days before any visible effects on the plants were observed (Barbagallo et al., 2003).
It is now possible to construct systems that are capable of imaging any parameter that can be measured with conventional (integrating) fluorometers. However, achieving this level of functionality in a cost-effective manner remains a significant challenge. Here, some important limitations of Chl a fluorescence imaging, and Chl a fluorometry in general, are considered. Attention is paid to the design limitations that are likely to be imposed by a limited budget. A more detailed description of the underlying technology of fluorescence imaging systems and a wider range of examples are provided in Oxborough (2004).
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