Two-photon absorption and excitation spectroscopy of carotenoids, chlorophylls and pigment-protein complexes

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden (dark) and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than similar to 600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

Automated summary

Studies have shown varying contributions of carotenoids and chlorophylls to the TPE/TPA spectra of pigment-protein complexes, with some reporting dominant TPE of carotenoids over chlorophyll a while others suggesting minor contributions of carotenoids. Collaboration between research teams is conducted to reconcile these discrepancies and it is concluded that carotenoids play a significant role in shorter wavelengths while chlorophylls dominate above 600/1200 nm in TPE spectra of LHCII and PSI.