No abstract

CP29, a chlorophyll (Chl) a/ b-xanthophyll binding protein, bridges energy transfer between the major LHCII antenna complexes and Photosystem II reaction centres. It hosts one of the two identified quenching sites making it crucial for regulated photoprotection mechanisms. CP29 photophysics has been so far studied on the purified protein in detergent solutions, since spectrally overlapping signals affect in vivo measurements. However, the protein in detergent assumes non-native conformations compared to its physiological state in the thylakoid membrane. Here, we report a detailed photophysical study on CP29 inserted in discoidal lipid bilayers, known as nanodiscs, which mimic the native membrane environment. Using picosecond time-resolved fluorescence and femtosecond transient absorption (TA), we observed shortening of the Chl fluorescence lifetime with a decrease of the carotenoid triplet formation yield for CP29 in nanodiscs as compared to the protein in detergent. Global analysis of TA data suggests a 1Chl* quenching mechanism dependent on excitation energy transfer to a carotenoid dark state, likely the proposed S*, which is believed to be formed due to a carotenoid conformational change affecting the S1 state. We suggest that the accessibility of the S* state in different local environments plays a key role in determining the quenching of Chl excited states. In vivo, non-photochemical quenching is activated by de-epoxidation of violaxanthin into zeaxanthin. CP29-Zeaxanthin in nanodiscs further shortens Chl lifetime, which underlines the critical role of zeaxanthin in modulating photoprotection activity.

Multispectral/hyperspectral Fluorescence Lifetime Imaging Microscopy (λFLIM) is a promising tool for studying functional and structural biological processes. The rich information content provided by a multidimensional dataset is often in contrast with the acquisition speed. In this work, we develop and experimentally demonstrate a wide-field λFLIM setup, based on a novel time-resolved x Single Photon Avalanche Diodes (SPAD) array detector working in a single pixel camera scheme, which parallelizes the spectral detection reducing the measurement time. The proposed system, which implements a singlepixel camera with compressive sensing scheme, represents an optimal microscopy framework towards the design of λFLIM setups.

Time-resolved fluorescence imaging is a key tool in biomedical applications, as it allows to non-invasively obtain functional and structural information. However, the big amount of collected data introduces challenges in both acquisition speed and processing needs. Here, we introduce a novel technique that allows to acquire a giga-voxel 4D hypercube in a fast manner while measuring only 0.03% of the dataset. The system combines two single-pixel cameras and a conventional 2D array detector working in parallel. Data fusion techniques are introduced to combine the individual 2D and 3D projections acquired by each sensor in the final high-resolution 4D hypercube, which can be used to identify different fluorophore species by their spectral and temporal signatures.