PAM measurement of fluorescence yield

The applied Multi-Color-PAM fluorometer like all PAM devices uses pulse-modulated ML and a special window-amplifier that is selective for the fluorescence excited by individual µs pulses of ML, so that the measurement of the ML-induced fluorescence is not disturbed by the fluorescence excited by much stronger AL or MT (Schreiber 1986, 2004). Hence, as ML-intensity is constant during measurements, the ML-excited fluorescence may be considered a measure of relative fluorescence yield that varies between a minimal value of Fo (dark-adapted sample, primary acceptor Q_A fully oxidized) and Fm (Q_A fully reduced in the absence of non-photochemical quenching). In contrast, fluorescence intensity may vary indefinitely, depending on the intensity of the applied non-modulated actinic illumination. The output of the Multi-Color-PAM is a voltage signal that can vary between 0 and 6 V. The amplitude of this signal not only depends on the relative fluorescence yield of the sample, but also on chlorophyll content, the ML-color, the chosen settings of ML-intensity and amplifier gain as well as on the choice of optical detector filters. Hence, while the signals always are proportional to fluorescence yield, the units with which the data are presented are arbitrary. In the present study, the instrument settings were generally optimized for maximal output signals of the various samples amounting to 4–6 V. The time-dependent fluorescence changes are plotted as relative fluorescence yield in arbitrary units, using the original voltage values for F < 710 signals and appropriately rescaled values for F > 700 signals (see Section below on "Rescaling for comparison of F > 700 and F < 710 data").