Technical features of a novel multi-color pulse amplitude modulation (PAM) chlorophyll fluorometer as well as the applied methodology and some typical examples of its practical application with suspensions of Chlorella vulgaris and Synechocystis PCC 6803 are presented. The multi-color PAM provides six colors of pulse-modulated measuring light (peak-wavelengths at 400, 440, 480, 540, 590, and 625 nm) and six colors of actinic light (AL), peaking at 440, 480, 540, 590, 625 and 420–640 nm (white). The AL can be used for continuous illumination, maximal intensity single-turnover pulses, high intensity multiple-turnover pulses, and saturation pulses. In addition, far-red light (peaking at 725 nm) is provided for preferential excitation of PS I. Analysis of the fast fluorescence rise kinetics in saturating light allows determination of the wavelength- and sample-specific functional absorption cross section of PS II, Sigma(II)λ, with which the PS II turnover rate at a given incident photosynthetically active radiation (PAR) can be calculated. Sigma(II)λ is defined for a quasi-dark reference state, thus differing from σPSII used in limnology and oceanography. Vastly different light response curves for Chlorella are obtained with light of different colors, when the usual PAR-scale is used. Based on Sigma(II)λ the PAR, in units of μmol quanta/(m2 s), can be converted into PAR(II) (in units of PS II effective quanta/s) and a fluorescence-based electron transport rate ETR(II) = PAR(II) · Y(II)/Y(II)max can be defined. ETR(II) in contrast to rel.ETR qualifies for quantifying the absolute rate of electron transport in optically thin suspensions of unicellular algae and cyanobacteria. Plots of ETR(II) versus PAR(II) for Chlorella are almost identical using either 440 or 625 nm light. Photoinhibition data are presented suggesting that a lower value of ETR(II)max with 440 nm possibly reflects photodamage via absorption by the Mn-cluster of the oxygen-evolving complex.

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A new type of computer controlled spectrophotometer is described which is based on an array of independent, monochromatic pulsed light sources consisting of light emitting diodes (LED) equipped with narrow band interference filters. The LEDs are sequentially pulsed at a high repetition rate. The absorbance information at specific wavelengths is sampled in the μs-time range, using a computer-controlled, highly selective technique of synchronous amplification. A first prototype of this LED Array Spectrophotometer allows simultaneous recording of kinetic changes at 16 different wavelengths in the range from 530 to 600 nm, with a time resolution of 1 ms/point. Special features of the new type of spectrophotometer are: Weak integrated measuring light intensity, high signal/noise ratio even with scattering samples like intact leaves, active baseline adjustment by LED current regulation, computer control of system operation and data analysis. To deconvolute the complex absorbance changes in the cytochrome α-band region, 'standard spectra' of the major components are stored in computer memory and used for curve fitting of difference spectra and kinetic changes. As an example of application, the light-induced absorbance changes in a heat-pretreated spinach leaf are analysed. The system effectively separates specific absorbance changes of C550, cyt f, cyt b 559 and cyt b 563 from a large background of non-specific changes.

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