Functional organization of 3D plant thylakoid membranes as seen by high resolution microscopy.

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.

Concentration Quenching of Fluorescence Decay Kinetics of Molecular Systems.

Fluorescence concentration quenching occurs when increasing molecular concentration of fluorophores results in a decreasing fluorescence quantum yield. Even though this phenomenon has been studied for decades, its mechanisms and signatures are not yet fully understood. The complexity of the problem arises due to energy migration and trapping in huge networks of molecules. Most of the available theoretical work focuses on integral quantities like fluorescence quantum yield and mean excitation lifetime. In this work, we present a numerical study of the fluorescence decay kinetics of three-dimensional and two-dimensional molecular systems. We investigate the differences arising from the variations in models of trap formations. We also analyze the influence of the molecular orientations to the fluorescence decay kinetics. We compare our results to the well-known analytical models and discuss their ranges of validity. Our findings suggest that the analytical models can provide inspiration for different ways of approximating the fluorescence kinetics, yet more detailed analysis of the experimental data should be done by comparison with numerical simulations.

Fluorescence quenching in aggregates of fucoxanthin-chlorophyll protein complexes: Interplay of fluorescing and dark states.

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.

Direct Tracking of Charge Carrier Drift and Extraction from Perovskite Solar Cells by Means of Transient Electroabsorption Spectroscopy.

The best perovskite solar cells currently demonstrate more than 25% efficiencies, yet many fundamental processes that determine the operation of these devices are still not fully understood. In particular, even though the device performance strongly depends on charge carrier transport across the perovskite layer to selective electrodes, information about this process is still very controversial. Here, we investigate charge carrier motion and extraction from an archetypical CH3NH3PbI3 (MAPI) perovskite solar cell. We use the ultrafast electric-field-modulated transient absorption technique, which allows us to evaluate the electric field dynamics from the time-resolved electroabsorption spectra and to visualize the motion of charge carriers with subpicosecond time resolution. We demonstrate that photogenerated holes drift across the mesoporous TiO2/perovskite layer during hundreds of picoseconds. On the other hand, their extraction into the spiro-OMeTAD hole transporting layer lasts for more than 1 nanosecond, suggesting that the hole extraction is limited by the perovskite/spiro-OMeTAD interface rather than by the hole transport through the perovskite layer. Additionally, we use the ultrafast time-resolved fluorescence technique that reveals fluorescence decay during tens of picoseconds, which we attribute to the spatial separation of electrons and holes.

Structure-based model of fucoxanthin-chlorophyll protein complex: Calculations of chlorophyll electronic couplings.

Diatoms are a group of marine algae that are responsible for a significant part of global oxygen production. Adapted to life in an aqueous environment dominated by the blue-green light, their major light-harvesting antennae-fucoxanthin-chlorophyll protein complexes (FCPs)-exhibit different pigment compositions than of plants. Despite extensive experimental studies, until recently the theoretical description of excitation energy dynamics in these complexes was limited by the lack of high-resolution structural data. In this work, we use the recently resolved crystallographic information of the FCP complex from Phaeodactylum tricornutum diatom [Wang et al., Science 363, 6427 (2019)] and quantum chemistry-based calculations to evaluate the chlorophyll transition dipole moments, atomic transition charges from electrostatic potential, and the inter-chlorophyll couplings in this complex. The obtained structure-based excitonic couplings form the foundation for any modeling of stationary or time-resolved spectroscopic data. We also calculate the inter-pigment Förster energy transfer rates and identify two quickly equilibrating chlorophyll clusters.

Quantum-Classical Approach for Calculations of Absorption and Fluorescence: Principles and Applications.

Absorption and fluorescence spectroscopy techniques provide a wealth of information on molecular systems. The simulations of such experiments remain challenging, however, despite the efforts put into developing the underlying theory. An attractive method of simulating the behavior of molecular systems is provided by the quantum-classical theory─it enables one to keep track of the state of the bath explicitly, which is needed for accurate calculations of fluorescence spectra. Unfortunately, until now there have been relatively few works that apply quantum-classical methods for modeling spectroscopic data. In this work, we seek to provide a framework for the calculations of absorption and fluorescence lineshapes of molecular systems using the methods based on the quantum-classical Liouville equation, namely, the forward-backward trajectory solution (FBTS) and the non-Hamiltonian variant of the Poisson bracket mapping equation (PBME-nH). We perform calculations on a molecular dimer and the photosynthetic Fenna-Matthews-Olson complex. We find that in the case of absorption, the FBTS outperforms PBME-nH, consistently yielding highly accurate results. We next demonstrate that for fluorescence calculations, the method of choice is a hybrid approach, which we call PBME-nH-Jeff, that utilizes the effective coupling theory [Gelzinis, A.; J. Chem. Phys. 2020, 152, 051103] to estimate the excited state equilibrium density operator. Thus, we find that FBTS and PBME-nH-Jeff are excellent candidates for simulating, respectively, absorption and fluorescence spectra of real molecular systems.

Confronting FCP structure with ultrafast spectroscopy data: evidence for structural variations.

Diatoms are a major group of algae, responsible for a quarter of the global primary production on our planet. Their adaptation to marine environments is ensured by their light-harvesting antenna - the fucoxanthin-chlorophyll protein (FCP) complex, which absorbs strongly in the blue-green spectral region. Although these essential proteins have been the subject of many studies, for a long time their comprehensive description was not possible in the absence of structural data. Last year, the 3D structures of several FCP complexes were revealed. The structure of an FCP dimer was resolved by crystallography for the pennate diatom Phaeodactylum tricornutum [W. Wang et al., Science, 2019, 363, 6427] and the structure of the PSII supercomplex from the centric diatom Chaetoceros gracilis, containing several FCPs, was obtained by electron microscopy [X. Pi et al., Science, 2019, 365, 6452; R. Nagao et al., Nat. Plants, 2019, 5, 890]. In this Perspective article, we evaluate how precisely these structures may account for previously published ultrafast spectroscopy results, describing the excitation energy transfer in the FCP from another centric diatom Cyclotella meneghiniana. Surprisingly, we find that the published FCP structures cannot explain several observations obtained from ultrafast spectroscopy. Using the available structures, and results from electron microscopy, we construct a trimer-based FCP model for Cyclotella meneghiniana, consistent with ultrafast experimental data. As a whole, our observations suggest that the structures from the proteins belonging to the FCP family display larger variations than the equivalent LHC proteins in plants, which may reflect species-specific adaptations or original strategies for adapting to rapidly changing marine environments.

Stark absorption and Stark fluorescence spectroscopies: Theory and simulations.

Stark spectroscopy experiments are widely used to study the properties of molecular systems, particularly those containing charge-transfer (CT) states. However, due to the small transition dipole moments and large static dipole moments of the CT states, the standard interpretation of the Stark absorption and Stark fluorescence spectra in terms of the Liptay model may be inadequate. In this work, we provide a theoretical framework for calculations of Stark absorption and Stark fluorescence spectra and propose new methods of simulations that are based on the quantum-classical theory. In particular, we use the forward-backward trajectory solution and a variant of the Poisson bracket mapping equation, which have been recently adapted for the calculation of conventional (field-free) absorption and fluorescence spectra. For comparison, we also apply the recently proposed complex time-dependent Redfield theory, while exact results are obtained using the hierarchical equations of motion approach. We show that the quantum-classical methods produce accurate results for a wide range of systems, including those containing CT states. The CT states contribute significantly to the Stark spectra, and the standard Liptay formalism is shown to be inapplicable for the analysis of spectroscopic data in those cases. We demonstrate that states with large static dipole moments may cause a pronounced change in the total fluorescence yield of the system in the presence of an external electric field. This effect is correctly captured by the quantum-classical methods, which should therefore prove useful for further studies of Stark spectra of real molecular systems. As an example, we calculate the Stark spectra for the Fenna-Matthews-Olson complex of green sulfur bacteria.

Light-harvesting complexes access analogue emissive states in different environments.

The light-harvesting complexes (LHCs) of plants can regulate the level of excitation in the photosynthetic membrane under fluctuating light by switching between different functional states with distinct fluorescence properties. One of the most fascinating yet obscure aspects of this regulation is how the vast conformational landscape of LHCs is modulated in different environments. Indeed, while in isolated antennae the highly fluorescent light-harvesting conformation dominates, LHC aggregates display strong fluorescence quenching, representing therefore a model system for the process of energy dissipation developed by plants to avoid photodamage in high light. This marked difference between the isolated and oligomeric conditions has led to the widespread belief that aggregation is the trigger for the photoprotective state of LHCs. Here, a detailed analysis of time-resolved fluorescence experiments performed on aggregates of CP29 - a minor LHC of plants - provides new insights into the heterogeneity of emissive states of this antenna. A comparison with the data on isolated CP29 reveals that, though aggregation can stabilize short-lived conformations to a certain extent, the massive quenching upon protein clustering is mainly achieved by energetic connectivity between complexes that maintain the same long-lived and dissipative states accessed in the isolated form. Our results also explain the typical far-red enhancement in the emission of antenna oligomers in terms of a sub-population of long-lived redshifted complexes competing with quenched complexes in the energy trapping. Finally, the role of selected chlorophylls in shaping the conformational landscape of the antenna is also addressed by studying mutants lacking specific pigments.

Benchmarking the forward-backward trajectory solution of the quantum-classical Liouville equation.

Various quantum-classical approaches to the simulation of processes taking place in real molecular systems have been shown to provide quantitatively correct results in a number of scenarios. However, it is not immediately clear how strongly the approximations related to the classical treatment of the system's environment compromise the accuracy of these methods. In this work, we present the analysis of the accuracy of the forward-backward trajectory solution (FBTS) of the quantum-classical Liouville equation. To this end, we simulate the excitation dynamics in a molecular dimer using the FBTS and the exact hierarchical equations of motion approach. To facilitate the understanding of the possible benefits of the FBTS, the simulations are also performed using a closely related quantum-classical Poisson Bracket Mapping Equation (PBME) method, as well as the well-known Förster and Redfield theories. We conclude that the FBTS is considerably more accurate than the PBME and the perturbative approaches for most realistic parameter sets and is, therefore, more versatile. We investigate the impact each parameter has on the accuracy of the FBTS. Our results can be used to predict whether the FBTS may be expected to yield satisfactory results when calculating system dynamics for the given system parameters.

Analytical derivation of equilibrium state for open quantum system.

Calculation of the equilibrium state of an open quantum system interacting with a bath remains a challenge to this day, mostly due to a huge number of bath degrees of freedom. Here, we present an analytical expression for the reduced density operator in terms of an effective Hamiltonian for a high temperature case. Comparing with numerically exact results, we show that our theory is accurate for slow baths and up to intermediate system-bath coupling strengths. Our results demonstrate that the equilibrium state does not depend on the shape of spectral density in the slow bath regime. The key quantity in our theory is the effective coupling between the states, which depends exponentially on the ratio of the reorganization energy to temperature and, thus, has opposite temperature dependence than could be expected from the small polaron transformation.

Aggregation-Related Nonphotochemical Quenching in the Photosynthetic Membrane.

The photosynthetic apparatus of plants is a robust self-adjustable molecular system, able to function efficiently under varying environmental conditions. Under strong sunlight, it switches into photoprotective mode to avoid overexcitation by safely dissipating the excess absorbed light energy via nonphotochemical quenching (NPQ). Unfortunately, heterogeneous organization and simultaneous occurrence of multiple processes within the thylakoid membrane impede the study of natural NPQ under in vivo conditions; thus, usually artificially prepared antennae have been studied instead. However, it has never been shown directly that the origin of fluorescence quenching observed in these artificial systems underlies natural NPQ. Here we report the time-resolved fluorescence measurements of the dark-adapted and preilluminated-to induce NPQ-intact chloroplasts, performed over a broad temperature range. We show that their spectral response matches that observed in the LHCII aggregates, thus demonstrating explicitly for the first time that the latter in vitro system preserves essential properties of natural photoprotection.

Two-dimensional spectroscopy for non-specialists.

Detailed studies of the excitation dynamics in photosynthetic pigment-proteins require an application of a wide range of spectroscopic methods. From the later part of the previous century, pump-probe and time-resolved fluorescence spectroscopy provided an impressive amount of information. Being simple to grasp, these methods are well-understood and widely used by the photosynthesis research community. In the last fifteen years, two-dimensional (2D) spectroscopy was developed. It has significant advantages over other methods, in particular higher temporal resolution available and higher signal-to-noise ratio. Even though it provides considerable opportunities in research, both its experimental realization and theoretical description are rather complicated, making it somewhat difficult to understand and apply. This makes an unfortunate gap in the community, with spectroscopy experts being able to use the technique, but sometimes lacking the relevant biological knowledge, while biologists having that knowledge are dubious about 2D spectroscopy due to the complexity of the approach. This publication is an attempt to fill this gap by providing an accessible introduction to the concepts, principles and possible applications of the 2D spectroscopy, aimed at the biologically trained members of the photosynthesis research community.

Can red-emitting state be responsible for fluorescence quenching in LHCII aggregates?

Non-photochemical quenching (NPQ) is responsible for protecting the light-harvesting apparatus of plants from damage at high light conditions. Although it is agreed that the major part of NPQ, an energy-dependent quenching (qE), originates in the light-harvesting antenna, its exact mechanism is still debated. In our earlier work (Chmeliov et al. in Nat Plants 2:16045, 2016), we have analyzed the time-resolved fluorescence (TRF) from the trimers and aggregates of the major light-harvesting complexes of plants (LHCII) over a broad temperature range and came to a conclusion that three distinct states are required to describe the experimental data: two of them correspond to the emission bands centered at ~680 and ~700 nm, and the third state is responsible for the excitation quenching. This was opposite to earlier suggestions of a two-state model, where the red-shifted fluorescence and excitation quenching were assumed to be related. To examine such possibility, in the current work we repeat our analysis of the TRF data in terms of the two-state model. We find that even though it can reasonably describe the aggregate fluorescence, it fails to do so for the LHCII trimers. We conclude that the red-emitting state cannot be responsible for fluorescence quenching in the LHCII aggregates and reaffirm that the three-state model is the simplest possible description of the experimental data.

Applicability of transfer tensor method for open quantum system dynamics.

Accurate simulations of open quantum system dynamics is a long standing issue in the field of chemical physics. Exact methods exist, but are costly, while perturbative methods are limited in their applicability. Recently a new black-box type method, called transfer tensor method (TTM), was proposed [J. Cerrillo and J. Cao, Phys. Rev. Lett. 112, 110401 (2014)]. It allows one to accurately simulate long time dynamics with a numerical cost of solving a time-convolution master equation, provided many initial system evolution trajectories are obtained from some exact method beforehand. The possible time-savings thus strongly depend on the ratio of total versus initial evolution lengths. In this work, we investigate the parameter regimes where an application of TTM would be most beneficial in terms of computational time. We identify several promising parameter regimes. Although some of them correspond to cases when perturbative theories could be expected to perform well, we find that the accuracy of such approaches depends on system parameters in a more complex way than it is commonly thought. We propose that the TTM should be applied whenever system evolution is expected to be long and accuracy of perturbative methods cannot be ensured or in cases when the system under consideration does not correspond to any single perturbative regime.

Spectroscopic properties of photosystem II reaction center revisited.

Photosystem II (PSII) is the only biological system capable of splitting water to molecular oxygen. Its reaction center (RC) is responsible for the primary charge separation that drives the water oxidation reaction. In this work, we revisit the spectroscopic properties of the PSII RC using the complex time-dependent Redfield (ctR) theory for optical lineshapes [A. Gelzinis et al., J. Chem. Phys. 142, 154107 (2015)]. We obtain the PSII RC model parameters (site energies, disorder, and reorganization energies) from the fits of several spectra and then further validate the model by calculating additional independent spectra. We obtain good to excellent agreement between theory and calculations. We find that overall our model is similar to some of the previous asymmetric exciton models of the PSII RC. On the other hand, our model displays differences from previous work based on the modified Redfield theory. We extend the ctR theory to describe the Stark spectrum and use its fit to obtain the parameters of a single charge transfer state included in our model. Our results suggest that ChlD1+PheoD1- is most likely the primary charge transfer state, but that the Stark spectrum of the PSII RC is probably also influenced by other states.

Ultrafast energy transfer within the photosystem II core complex.

We report 2D electronic spectroscopy on the photosystem II core complex (PSII CC) at 77 K under different polarization conditions. A global analysis of the high time-resolution 2D data shows rapid, sub-100 fs energy transfer within the PSII CC. It also reveals the 2D spectral signatures of slower energy equilibration processes occurring on several to hundreds of picosecond time scales that are consistent with previous work. Using a recent structure-based model of the PSII CC [Y. Shibata, S. Nishi, K. Kawakami, J. R. Shen and T. Renger, J. Am. Chem. Soc., 2013, 135, 6903], we simulate the energy transfer in the PSII CC by calculating auxiliary time-resolved fluorescence spectra. We obtain the observed sub-100 fs evolution, even though the calculated electronic energy shows almost no dynamics at early times. On the other hand, the electronic-vibrational interaction energy increases considerably over the same time period. We conclude that interactions with vibrational degrees of freedom not only induce population transfer between the excitonic states in the PSII CC, but also reshape the energy landscape of the system. We suggest that the experimentally observed ultrafast energy transfer is a signature of excitonic-polaron formation.

The nature of self-regulation in photosynthetic light-harvesting antenna.

The photosynthetic apparatus of green plants is well known for its extremely high efficiency that allows them to operate under dim light conditions. On the other hand, intense sunlight may result in overexcitation of the light-harvesting antenna and the formation of reactive compounds capable of 'burning out' the whole photosynthetic unit. Non-photochemical quenching is a self-regulatory mechanism utilized by green plants on a molecular level that allows them to safely dissipate the detrimental excess excitation energy as heat. Although it is believed to take place in the plant's major light-harvesting complexes (LHC) II, there is still no consensus regarding its molecular nature. To get more insight into its physical origin, we performed high-resolution time-resolved fluorescence measurements of LHCII trimers and their aggregates across a wide temperature range. Based on simulations of the excitation energy transfer in the LHCII aggregate, we associate the red-emitting state, having fluorescence maximum at ∼700 nm, with the partial mixing of excitonic and chlorophyll-chlorophyll charge transfer states. On the other hand, the quenched state has a totally different nature and is related to the incoherent excitation transfer to the short-lived carotenoid excited states. Our results also show that the required level of photoprotection in vivo can be achieved by a very subtle change in the number of LHCIIs switched to the quenched state.

Coherence and population dynamics of chlorophyll excitations in FCP complex: Two-dimensional spectroscopy study.

Energy transfer processes and coherent phenomena in the fucoxanthin-chlorophyll protein complex, which is responsible for the light harvesting function in marine algae diatoms, were investigated at 77 K by using two-dimensional electronic spectroscopy. Experiments performed on femtosecond and picosecond timescales led to separation of spectral dynamics, witnessing evolutions of coherence and population states of the system in the spectral region of Qy transitions of chlorophylls a and c. Analysis of the coherence dynamics allowed us to identify chlorophyll (Chl) a and fucoxanthin intramolecular vibrations dominating over the first few picoseconds. Closer inspection of the spectral region of the Qy transition of Chl c revealed previously not identified, mutually non-interacting chlorophyll c states participating in femtosecond or picosecond energy transfer to the Chl a molecules. Consideration of separated coherent and incoherent dynamics allowed us to hypothesize the vibrations-assisted coherent energy transfer between Chl c and Chl a and the overall spatial arrangement of chlorophyll molecules.

Absorption lineshapes of molecular aggregates revisited.

Linear absorption is the most basic optical spectroscopy technique that provides information about the electronic and vibrational degrees of freedom of molecular systems. In simulations of absorption lineshapes, often diagonal fluctuations are included using the cumulant expansion, and the off-diagonal fluctuations are accounted for either perturbatively, or phenomenologically. The accuracy of these methods is limited and their range of validity is still questionable. In this work, a systematic study of several such methods is presented by comparing the lineshapes with exact results. It is demonstrated that a non-Markovian theory for off-diagonal fluctuations, termed complex time dependent Redfield theory, gives good agreement with exact lineshapes over a wide parameter range. This theory is also computationally efficient. On the other hand, accounting for the off-diagonal fluctuations using the modified Redfield lifetimes was found to be inaccurate.