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Combined contributions of carotenoids and chlorophylls in two-photon spectra of photosynthetic pigment-protein complexes-A new way to quantify carotenoid dark state to chlorophyll energy transfer?

Transitions into the first excited state of carotenoids, Car S1, are optically forbidden in conventional one-photon excitation (OPE) but are possible via two-photon excitation (TPE). This can be used to quantify the amount of Car S1 to Chlorophyll (Chl) energy transfer in pigment-protein complexes and plants by observing the chlorophyll fluorescence intensity after TPE in comparison to the intensity observed after direct chlorophyll OPE. A parameter, ΦCoupling Car S1-Chl, can be derived that directly reflects relative differences or changes in the Car S1 → Chl energy transfer of different pigment-protein complexes and even living plants. However, very careful calibrations are necessary to ensure similar OPE and TPE excitation probabilities and transition energies. In plants, the exact same sample spot must be observed at the same time. All this is experimentally quite demanding. ΦCoupling Car S1-Chl also corrects intrinsically for direct chlorophyll TPE caused by larger chlorophyll excesses in the complexes, but recently it turned out that in certain TPE wavelengths ranges, its contribution can be quite large. Fortunately, this finding opens also the possibility of determining ΦCoupling Car S1-Chl in a much easier way by directly comparing values in TPE spectra observed at wavelengths that are either more dominated by Cars or Chls. This avoids tedious comparisons of OPE and TPE experiments and potentially allows measurement at even only two TPE wavelengths. Here, we explored this new approach to determine ΦCoupling Car S1-Chl directly from single TPE spectra and present first examples using known experimental spectra from Cars, Chl a, Chl b, LHC II, and PS 1.