Devices and methods for room-temperature fluorescence analysis

An overview of the various types of photochemical and non-photochemical fluorescence quenching in vivo is given. Devices and methods are outlined that allow specific information to be obtained from complex fluorescence responses. The importance of correlated measurements of other, independent photosynthesis signals is emphasized. It is shown that a recently introduced pulse-amplitude modulation fluorometer (PAM fluorometer) can also be used with modified emitter-detector units to measure absorbance changes. Examples are given for absorbance changes of the Hill reagent methyl purple, induced by single turnover flashes, and for P700 absorbance changes measured simultaneously with fluorescence. Correlated P700 and fluorescence measurements give deeper insights into the control of electron transfer from PQH2 to cytochrome (cyt) b/f and into the intersystem acceptor-pool size of sun and shade leaves. Possible explanations for differences in pool sizes determined by P700 and fluorescence measurements are discussed. By using P700 reduction as an indicator, it is shown that in saturating light the plastoquinone (PQ) pool is already reduced within 50 ms, whereas the last phase of the fluorescence rise (I2-P) takes about 300 ms and is paralleled by the re-reduction of P700. It is concluded that I2-P reflects removal of photochemical quenching at PSI and that 50 ms saturation pulses are appropriate to eliminate the relevant photochemical PSII quenching.