Carotenoid molecules are critical in photosynthesis, performing functions at the heart of both light harvesting and photoprotection. As both these processes involve excitation energy transfer, fully understanding them requires a precise description of the electronic states involved. The excited-state manifold of carotenoids is not yet fully characterized and includes several dark electronic states that remain elusive. Using femtosecond-stimulated resonance Raman spectroscopy, where the vibrational contributions of each excited state can be observed selectively as a function of the Raman excitation, we resolve vibrational signatures consistent with three dark-state contributions and propose assignments for them. These results address long-standing controversies in carotenoid research and provide a spectroscopic framework that is relevant to the multiple roles of these molecules.

Photosynthetic light-harvesting complexes (LHCs) exhibit remarkable diversity in their electronic properties despite structural homology. In this study, we investigate the environment-dependent local excitations and chlorophyll-chlorophyll charge transfer (CT) states in the LHCI antenna of plant Photosystem I, focusing on the Lhca1-4 subcomplexes. Using high-resolution structural data from Mazor et al. [Nat. Plants, 3, 17014 (2017); PDB ID: 5L8R], we performed QM/MM calculations to quantify electrostatic energy shifts induced by the protein environment. We identified seven low energy states with non-negligible CT mixing that may contribute to the red-shifted fluorescence observed experimentally. Comparative analysis with previous results based on the slightly lower resolution structure by Qin et al. [Science, 348, 989 (2015); PDB ID: 4XK8] reveals significant sensitivity of CT state energetics to subtle structural variations and protonation patterns of the amino acids, particularly involving GLU153 in Lhca4. Our findings underscore the critical importance of accurate structural modeling and protonation state assignment in predicting pigment electronic properties and interpreting spectroscopic data. This work advances our understanding of the mechanistic basis of red-shifted fluorescence in LHCI and highlights the need for multi-structure validation in theoretical studies of photosynthetic complexes.

Lutein is a xanthophyll carotenoid with a conjugated system terminated by two ionone rings, one of which extends the π-conjugation. Its photophysical behaviour in different environments is poorly understood. Here, we report room-temperature excited-state characterisation of lutein in two solvents with strongly different polarisabilities, THF and CS2, combining femtosecond transient absorption and femtosecond stimulated resonance Raman spectroscopy. In both environments, we resolve the same set of excited states, including a hot S1 state, relaxed S1, a weak ICT contribution coupled to S1, and a longer-lived dark state with triplet-pair character, S*. Despite this shared state manifold, the two solvents produce markedly different behaviours. In CS2, the S1 absorption band is substantially red-shifted and broadened relative to THF, and the vibrational dynamics of the excited-state manifold evolve much less over time, indicating that highly polarisable environments suppress relaxation-driven spectral evolution. The S* dark state lifetime of ∼16 ps in THF increases to approximately 29 ps in CS2, and its vibrational frequency shifts upward by 8 cm-1, deviating from the scaling observed for linear carotenoids of comparable conjugation length.

Photosynthetic antenna proteins harvest light energy while at the same time protecting the organism against photodamage. Carotenoid molecules are essential in the latter process, efficiently quenching unwanted (bacterio)chlorophyll excited states created after photon absorption. (Bacterio)chlorophyll triplets, formed by inter-system crossing, are particularly significant, since in the absence of carotenoid quenching, they sensitise the highly oxidative singlet oxygen. In light-harvesting complex 2 (LH2) from Rhodoblastus acidophilus, the pathways that populate carotenoid dark and triplet states remain controversial, involving bacteriochlorophyll-to-carotenoid triplet-triplet transfer and/or generation of triplets by the carotenoid molecules themselves through singlet fission. Transient absorption has been central to understanding photoprotection in these organisms, but spectral congestion limits the separation of the overlapping species needed to discriminate between these pathways. By applying femtosecond stimulated resonance Raman spectroscopy (FSRRS) in different resonance conditions to this protein, in combination with an extension of global analysis to four dimensions (wavenumber, time, intensity and resonance condition), we separate each component of the carotenoid dark-state manifold together with its kinetics. An entangled triplet pair S*/1(TT) is observed, which lives about 60 ps, some eight times longer than in solution. However, this stabilisation does not open a pathway to separated triplets or to carotenoid-BChl a heterofission. Triplet-triplet transfer from bacteriochlorophyll a to carotenoid is also resolved under BChl a excitation, and fits cleanly as a single 2100 ps component. The carotenoid triplet in LH2 is thus produced by photoprotective triplet-triplet transfer from BChl a, and not by singlet fission.

Vibrational spectroscopy reports on molecular structure with chemical-bond specificity, but in biological systems the vibrational signals of a target chromophore are typically buried under contributions from the surrounding matrix. Resonance Raman (RR) spectroscopy addresses this problem by matching the excitation wavelength to an electronic transition of the chromophore of interest, which increases Raman cross sections by up to six orders of magnitude and restricts the enhanced modes to those coupled to the resonant electronic state. This chapter introduces the physical basis of resonance enhancement and shows how RR isolates chromophore-specific vibrational markers in complex biological systems. We then extend the same principle into the time domain with femtosecond stimulated resonance Raman spectroscopy (FSRRS). In FSRRS, the Raman pump is tuned across the visible range and can be placed in resonance with the transient absorption of a chosen excited-state species, which makes the Raman pump wavelength an additional experimental variable. Sampling this variable across the excited-state absorption manifold provides a selection criterion that separates coexisting transient species sharing a common vibrational window.

Light-harvesting and excitation energy transfer in photosynthesis generally involve chlorophyll-molecules, maintained by their host proteins at short distances from each other, this resulting in excitonic coupling. The transfer of excitation energy to the reaction centers consists of exciton migration and relaxation within and between photosynthetic proteins. The dynamics of this process depends on the vibrational modes resonant with the energy gaps between the participating excited states. The precise structure and vibrational landscape of excitons is thus essential knowledge to understand the amazing efficiency of photosynthesis. In this work, we characterize the vibrational properties of excitons in light-harvesting proteins from purple photosynthetic bacteria, which remarkably unveil on how many bacteriochlorophylls they reside and in which proportions. Vibrational spectra obtained from bacteriochlorophylls in proteins generally contain additional vibronic contributions when compared to isolated pigments, opening additional pathways for vibrationally-assisted excitation energy transfer. In contrast, the absence of new vibronic contributions in the spectra of chlorophyll -containing photosynthetic proteins above 100 cm-1 suggests that in oxygenic photosynthesis, vibrationally-assisted excitation energy transfers occurs through vibrational modes of chlorophyll molecules in equilibrium configuration.

Desiccation-tolerant mosses survive extreme water loss by activating efficient photoprotective mechanisms that prevent damage to the photosynthetic apparatus. The most resistant moss Syntrichia caninervis (S. caninervis) represents an important model for studying the molecular basis of dehydration resilience; however, the membrane and protein level processes, which enable rapid recovery of photosynthetic activity upon rehydration, remain insufficiently understood. In this work, we investigate the fluorescence (FL) dynamics of the photosynthetic apparatus of S. caninervis under three hydration states (hydrated, dehydrated and desiccated) in whole leaves using time-resolved FL spectroscopy. Time-resolved measurements with 10 ps resolution were performed to resolve excitation dynamics in both photosystems I (PSI) and II (PSII). The complex FL datasets were analysed using the spectral decomposition approach. Dehydrated and desiccated samples exhibit substantially reduced FL intensity compared to hydrated moss in steady-state experiments, indicating efficient quenching of chlorophyll excitation upon drying. The time-resolved FL measurements revealed that excitation transfer rates and the nature of quenched states strongly depend on hydration level in S. caninervis. In the hydrated state, FL dynamics are consistent with functional photosystems in plants. Upon dehydration, non-photochemical quenching is activated in both PSII and PSI, with PSII quenching persisting even at low temperatures. We therefore conclude, that S. caninervis employs few hydration-dependent photoprotective regimes, including distinct quenching mechanisms at both photosystems. Our results suggest the presence of ultrafast quenching processes under severe dehydration, which are progressively relieved during rehydration and replaced by alternative mechanisms supporting the recovery of photosynthetic activity.

Diatoms are unicellular photosynthetic microalgae that contribute nearly a quarter of the global primary production. Knowledge of the mechanisms, networks, and time scales of the excitation transfer is crucial for a deeper understanding of their photosynthetic machinery. Unfortunately, the short excited-state lifetime of photosystem I at room temperature and research focus on isolated structures have limited the development of a complete excitation transfer scheme in intact cells. Here, we address this gap through picosecond time-resolved fluorescence measurements on intact Cyclotella meneghiniana cells obtained from 16 to 130 K and room temperature. Analysis of the data in terms of evolution-associated spectra reveals a complex interplay of excitation transfer, population of fluorescing trapping states, and excitation quenching at the whole-cell level. We show rapid depopulation of all antenna complexes, on the order of 30 ps even at 16 K, indicating a more efficient energy-transfer network in diatoms than in higher plants.

We have prepared lycopene aggregates with low scattering in an acetone-water suspension. The aggregates exhibit highly distorted absorption, extending from the UV up to 568 nm, as a result of strong excitonic interactions. We have investigated the structural organization of these aggregates by resonance Raman and TEM, revealing that the lycopene aggregates are not homogeneous, containing at least five different aggregate species. Transient absorption measurements upon excitation at 355, 515, and 570 nm, to sub-select these different species, reveal significant differences in dynamics between each of the aggregate types. The strong excitonic interactions produce highly distorted transient electronic signatures, which do not allow an unequivocal identification of the excited states at times shorter than 60 ps. However, these experiments demonstrate that all the lycopene aggregated species form long-living triplets via singlet fission.

We provide the first direct evidence of singlet fission occurring with water-soluble compounds. We show that perylene-3,4,9,10-tetracarboxylate forms dynamic dimers in aqueous solution, with lifetimes long enough to allow intermolecular processes such as singlet fission. As these are transient dimers rather than stable aggregates, they retain a significant degree of disorder. We performed a comprehensive analysis of such dynamic assemblies using time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, and theoretical modelling, allowing us to observe the characteristic signatures of singlet fission and develop a model to characterize the different species observed. Our findings reveal that structure fluctuations within perylene associations are key in favoring either singlet fission or charge separation. The efficiency of triplet formation is higher than 100%, and the disordered system leads to triplets living in the nanosecond time range.

Light nanoscopy is attracting widespread interest for the visualization of fluorescent structures at the nanometer scale, especially in cellular biology. To achieve nanoscale resolution, one has to surpass the diffraction limit—a fundamental phenomenon determining the spot size of focused light. Recently, a variety of methods have overcome this limit, yet in practice they are often constrained by the requirement of special fluorophores, nontrivial data processing, or high price and complex implementation. For this reason, confocal fluorescence microscopy that yields relatively low resolution is still the dominant method in biomedical sciences. It was shown that image scanning microscopy (ISM) with an array detector instead of a point detector could improve the resolution of confocal microscopy. Here we review the principles of the confocal microscopy and present a simple method based on ISM with a different image reconstruction approach, which can be easily implemented in any camera-based laser-scanning set-up to experimentally obtain the theoretical resolution limit of the confocal microscopy. Our method, Single Pixel Reconstruction Imaging (SPiRI) enables high-resolution 3D imaging utilizing image formation only from a single pixel of each of the recorded frames. We achieve experimental axial resolution of 330 nm, which was not shown before by basic confocal or ISM-based systems. Contrary to the majority of techniques, SPiRI method exhibits a low lateral-to-axial FWHM aspect ratio, which means a considerable improvement in 3D fluorescence imaging of cellular structures. As a demonstration of SPiRI application in biomedical sciences, we present a 3D structure of bacterial chromosome with excellent precision.

We provide direct evidence of singlet fission occurring with water-soluble compounds. We show that perylene-3,4,9,10-tetracarboxylate forms dynamic dimers in aqueous solution, with lifetimes long enough to allow intermolecular processes such as singlet fission. As these are transient dimers rather than stable aggregates, they retain a significant degree of disorder. We performed a comprehensive analysis of such dynamic assemblies using time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, and theoretical modelling, allowing us to observe the characteristic signatures of singlet fission and develop a model to characterize the different species observed. Our findings reveal that structure fluctuations within perylene-3,4,9,10-tetracarboxylate associations are key in favoring either singlet fission or charge separation. The efficiency of triplet formation is higher than 100%, and the disordered system leads to triplets living in the nanosecond time range.

In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

Photosystem I (PSI) light-harvesting antenna complexes LHCI contain spectral forms that absorb and emit photons of lower energy than that of its primary electron donor, P700. The most red-shifted fluorescence is associated with the Lhca4 complex. It has been suggested that this red emission is related to the inter-chlorophyll charge transfer (CT) states. In this work we present a systematic quantum-chemical study of the CT states in Lhca4, accounting for the influence of the protein environment by estimating the electrostatic interactions. We show that significant energy shifts result from these interactions and propose that the emission of the Lhca4 complex is related not only to the previously proposed a603+-a608- state, but also to the a602+-a603- state. We also investigate how different protonation patterns of protein amino acids affect the energetics of the CT states.

Correction for ‘Perylene-derivative singlet exciton fission in water solution’ by Chloe Magne et al., Chem. Sci., 2024, 15, 17831–17842, https://doi.org/10.1039/D4SC04732J.

In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL, HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to.

Diatoms, a major group of algae, account for about a quarter of the global primary production on Earth. These photosynthetic organisms face significant challenges due to light intensity variations in their underwater habitat. To avoid photodamage, they have developed very efficient non -photochemical quenching (NPQ) mechanisms. These mechanisms originate in their light -harvesting antenna - the fucoxanthin-chlorophyll protein (FCP) complexes. Spectroscopic studies of NPQ in vivo are often hindered by strongly overlapping signals from the photosystems and their antennae. Fortunately, in vitro FCP aggregates constitute a useful model system to study fluorescence (FL) quenching in diatoms. In this work, we present streak -camera FL measurements on FCPa and FCPb complexes, isolated from a centric diatom Cyclotella meneghiniana, and their aggregates. We find that spectra of non -aggregated FCP are dominated by a single fluorescing species, but the FL spectra of FCP aggregates additionally contain contributions from a redshifted emissive state. We relate this red state to a charge transfer state between chlorophyll c and chlorophyll a molecules. The FL quenching, on the other hand, is due to an additional dark state that involves incoherent energy transfer to the fucoxanthin carotenoids. Overall, the global picture of energy transfer and quenching in FCP aggregates is very similar to that of major light -harvesting complexes in higher plants (LHCII), but microscopic details between FCPs and LHCIIs differ significantly.

We provide the first direct evidence of singlet fission occurring with water-soluble compounds. Perylene-3,4,9,10-tetracarboxylic forms dynamic oligomers in aqueous solution, with lifetimes long enough to allow intermolecular processes such as singlet fission. As these are transient oligomers rather than large, stable aggregates, they retain a significant degree of disorder. We performed a comprehensive analysis of such dynamic assemblies using time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, and theoretical modelling, allowing us to observe the characteristic signatures of singlet fission and develop a model to explain the different species observed. Our findings reveal that the twist and tilt angles between perylenes are key in favoring either singlet fission or charge separation. The efficiency of triplet formation is higher than 100% and the disordered system leads to triplets living in the nanosecond time range.

Carotenoids are conjugated linear molecules built from the repetition of terpene units, which display a large structural diversity in nature. They may, in particular, contain several types of side or end groups, which tune their functional properties, such as absorption position and photochemistry. We report here a detailed experimental study of the absorption and vibrational properties of allene-containing carotenoids, together with an extensive modeling of these experimental data. Our calculations can satisfactorily explain the electronic properties of vaucheriaxanthin, where the allene group introduces the equivalent of one C=C double bond into the conjugated C=C chain. The position of the electronic absorption of fucoxanthin and butanoyloxyfucoxanthin requires long-range corrections to be found correctly on the red side of that of vaucheriaxanthin; however, these corrections tend to overestimate the effect of the conjugated and nonconjugated C=O groups in these molecules. We show that the resonance Raman spectra of these carotenoids are largely perturbed by the presence of the allene group, with the two major Raman contributions split into two components. These perturbations are satisfactorily explained by modeling, through a gain in the Raman intensity of the C=C antisymmetric stretching mode, induced by the presence of the allene group in the carotenoid C=C chain.

Resonance Raman spectroscopy is one of the most powerful techniques in analytical science due to its molecular selectivity, high sensitivity, and the fact that, in contrast to IR absorption spectroscopy, the presence of water does not hamper or mask the results. Originating in physics and chemistry, the use of Raman spectroscopy has spread and now includes a variety of applications in different disciplines, including biology. In this chapter, we introduce the basic principles of Raman and resonance Raman scattering, and show resonance Raman can be applied to study carotenoid molecules, in complex biological or chemical matrices. We describe the type of information that can be extracted from resonance Raman spectra, illustrating the power of this method by a series of example applications.