FTM displayed in Fig. 2 may serve to outline some points essential for optimal measurements of the O–I 1 rise kinetics: (1) The pulse-modulated fluorescence ML is switched
on only 100 μs before onset of AL to minimize the fluorescence rise induced by the ML and, hence, to allow use of relatively high ML-intensity setting for the sake of a high signal/noise ratio. (2) Maximal measuring pulse-frequency (MFmax) is triggered simultaneously with ML-on. The default setting of MFmax = 100 kHz provides sufficient selleckchem time resolution for reliable assessment of the O–I 1 kinetics with time constants in the order of 200 μs. (3) AL is triggered at time −5 μs to take account of a small time delay between switching of the AL-LED-driver and AL-on. (4) The amplifier “gating” (S&H off) is triggered on for 15 μs for AL-on (from −10 to 5 μs) and for 80 μs for the 50 μs ST pulse (from 995 to 1,075 μs). Consecutive measurements of O–I 1 rise kinetics driven by strong 440-, 480-, 540-, 590-, and 625-nm light of the same sample were preprogrammed in special Script-files for Chlorella and Synechocystis with 10-s dark-time between measurements. For each color, ML-intensity/Gain settings were programmed to
give approximately equal F o values. AL/MT-intensity settings were programmed such that for the investigated organism the initial rise curves displayed similar slopes with all the colors. Analysis of O–I 1 rise kinetics The eFT508 kinetics of the O–I 1 fluorescence rise were analyzed with the help of a dedicated fitting routine developed for determination of the wavelength-dependent absorption cross section of PS II, here called Sigma(II)λ. Fitting is based on Adenylyl cyclase the reversible radical
pair model of PS II originally described by Lavergne and Trissl (1995) that was extended to take account of Q A − -find more reoxidation (Klughammer C, Kolbowski J and Schreiber U, in preparation). Variable parameters in this model, fitted by the PamWin-3 program, are: J Sigmoidicity parameter, which is related to Joliot’s connectivity parameter, p, via the equation J = p/(1 – p) Tau Time constant of light-driven reduction of QA (by AL or MT pulse), corresponding to the inverse of the rate constant of PS II turnover, k(II) Tau(reox) Time constant of Q A − -reoxidation. Directly measured parameters are the F o and I 1-levels, which define the total range of ∆F that can be induced by a saturating ST flash (ST pulse) in the presence of an oxidized PQ-pool. The fitted parameters refer to the kinetics of QA-reduction, i.e., the increase of (1 − q), where q represents the fraction of open PS II reaction centers.