2008), this would allow to have ~100% of the RCs open in the streak-camera experiment described above, even if PSI was reduced at a rate of 4/s. Such a reduction rate can be obtained using 1 μM of PMS, which will not notably quench the fluorescence (Bulychev and Vredenberg 2001). The special spinning cuvette also allows performing transient absorption (Müller et al. 2003; Holzwarth et al. 2006) and TCSPC (Slavov et al. 2008) experiments with nearly all the PSI RCs in open state. Another obvious

solution to lower the fraction of closed RCs is to lower the excitation power. For a very sensitive technique, for example TCSPC, this can still give data with a good signal to noise ratio. However, for the other techniques such as fluorescence up-conversion, this will not be possible, and one might have to settle with measuring PSI with closed AR-13324 in vivo RCs (Kennis et al. 2001). PMS: to add or not to add? Our study shows that the commonly used reducing agent PMS quenches the fluorescence emission of PSI. This effect might be avoided using very low concentrations of PMS (Bulychev and Vredenberg 2001), but under this condition

the P700 reduction rate is also low. Another disadvantage of PMS is its low stability in water. Decomposition of solutions in deionized water takes only hours, while the stability is even lower in neutral JIB04 mw buffers (Sigma Product Information sheet). Thus, during long measurements the actual PMS concentration, and thus the P700 reduction rate, will be lower than expected. The best solution would be to find a stable and fast P700 reducing agent that does not quench chlorophyll fluorescence. In the absence of such a reagent it can be preferable, depending on the goal PIK3C2G of the experiment, to measure PSI with closed RCs as the fluorescence quantum yield and thus the trapping efficiency is only slightly dependent on the P700 oxidative state (Fig. 5). Acknowledgments This study was supported

by the De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO), Earth and Life Sciences (ALW), through a Vidi grant (to R.C.). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Amunts A, Toporik H, Borovikova A, Nelson N (2010) Structure determination and improved model of plant photosystem I. J Biol Chem 285:3478–3486PubMedCrossRef Bassi R, Simpson D (1987) Chlorophyll-protein complexes of barley photosystem-I. Eur J Biochem 163:221–learn more 230PubMedCrossRef Ben-Shem A, Frolow F, Nelson N (2003) Crystal structure of plant photosystem I. Nature 426:630–635PubMedCrossRef Berthold DA, Babcock GT, Yocum CF (1981) A highly resolved, oxygen-evolving photosystem-II preparation from spinach thylakoid membranes—electron-paramagnetic-res and electron-transport properties.