Site-Directed Mutagenesis of the Chlorophyll-Binding Sites Modulates Excited-State Lifetime and Chlorophyll-Xanthophyll Energy Transfer in the Monomeric Light-Harvesting Complex CP29.

We combine site-directed mutagenesis with picosecond time-resolved fluorescence and femtosecond transient absorption (TA) spectroscopies to identify excitation energy transfer (EET) processes between chlorophylls (Chls) and xanthophylls (Xant) in the minor antenna complex CP29 assembled inside nanodiscs, which result in quenching. When compared to WT CP29, a longer lifetime was observed in the A2 mutant, missing Chl a612, which closely interacts with Xant Lutein in site L1. Conversely, a shorter lifetime was obtained in the A5 mutant, in which the interaction between Chl a603 and Chl a609 is strengthened, shifting absorption to lower energy and enhancing Chl-Xant EET. Global analysis of TA data indicated that EET from Chl a Qy to a Car dark state S* is active in both the A2 and A5 mutants and that their rate constants are modulated by mutations. Our study provides experimental evidence that multiple Chl-Xant interactions are involved in the quenching activity of CP29.

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing.

Significance: The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. Aim: To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. Approach: A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. Results: The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. Conclusions: An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.

Influence of structural colour on the photoprotective mechanism in the gametophyte phase of the red alga Chondrus crispus.

Marine life is populated by a huge diversity of organisms with an incredible range of colour. While structural colour mechanisms and functions are usually well studied in marine animal species, there is a huge knowledge gap regarding the marine macroalgae (red, green and brown seaweeds) that have structural coloration and the biological significance of this phenomenon in these photosynthetic organisms. Here we show that structural colour in the gametophyte life history phase of the red alga Chondrus crispus plays an important role as a photoprotective mechanism in synergy with the other pigments present. In particular, we have demonstrated that blue structural coloration attenuates the more energetic light while simultaneously favouring green and red light harvesting through the external antennae (phycobilisomes) which possess an intensity-dependent photoprotection mechanism. These insights into the relationship between structural colour and photosynthetic light management further our understanding of the mechanisms involved.

Enhancement of Photoluminescence Properties via Polymer Infiltration in a Colloidal Photonic Glass.

Photonic glasses (PGs) based on the self-assembly of monosized nanoparticles can be an effective tool for realizing disordered structures capable of tailoring light diffusion due to the establishment of Mie resonances. In particular, the wavelength position of these resonances depends mainly on the morphology (dimension) and optical properties (refractive index) of the building blocks. In this study, we report the fabrication and optical characterization of photonic glasses obtained via a self-assembling technique. Furthermore, we have demonstrated that the infiltration of these systems with a green-emitting polymer enhances the properties of the polymer, resulting in a large increase in its photoluminescence quantum yield and a 3 ps growing time of the photoluminescence time decay Finally, the development of the aforementioned system can serve as a suitable low-cost platform for the realization of lasers and fluorescence-based bio-sensors.

Fast data fitting scheme for compressive multispectral fluorescence lifetime imaging.

A single-pixel camera combined with compressive sensing techniques is a promising fluorescence microscope scheme for acquiring a multidimensional dataset (space, spectrum, and lifetime) and for reducing the measurement time with respect to conventional microscope schemes. However, upon completing the acquisition, a computational step is necessary for image reconstruction and data analysis, which can be time-consuming, potentially canceling out the beneficial effect of compressive sensing. In this work, we propose and experimentally validate a fast-fit workflow based on global analysis and multiple linear fits, which significantly reduces the computation time from tens of minutes to less than 1 s. Moreover, as the method is interlaced with the measurement flow, it can be applied in parallel with the acquisitions.

Electronic Structure of Isolated Graphene Nanoribbons in Solution Revealed by Two-Dimensional Electronic Spectroscopy.

Structurally well-defined graphene nanoribbons (GNRs) are nanostructures with unique optoelectronic properties. In the liquid phase, strong aggregation typically hampers the assessment of their intrinsic properties. Recently we reported a novel type of GNRs, decorated with aliphatic side chains, yielding dispersions consisting mostly of isolated GNRs. Here we employ two-dimensional electronic spectroscopy to unravel the optical properties of isolated GNRs and disentangle the transitions underlying their broad and rather featureless absorption band. We observe that vibronic coupling, typically neglected in modeling, plays a dominant role in the optical properties of GNRs. Moreover, a strong environmental effect is revealed by a large inhomogeneous broadening of the electronic transitions. Finally, we also show that the photoexcited bright state decays, on the 150 fs time scale, to a dark state which is in thermal equilibrium with the bright state, that remains responsible for the emission on nanosecond time scales.

Time domain diffuse Raman spectroscopy using single pixel detection.

Diffuse Raman spectroscopy (DIRS) extends the high chemical specificity of Raman scattering to in-depth investigation of thick biological tissues. We present here a novel approach for time-domain diffuse Raman spectroscopy (TD-DIRS) based on a single-pixel detector and a digital micromirror device (DMD) within an imaging spectrometer for wavelength encoding. This overcomes the intrinsic complexity and high cost of detection arrays with ps-resolving time capability. Unlike spatially offset Raman spectroscopy (SORS) or frequency offset Raman spectroscopy (FORS), TD-DIRS exploits the time-of-flight distribution of photons to probe the depth of the Raman signal at a single wavelength with a single source-detector separation. We validated the system using a bilayer tissue-bone mimicking phantom composed of a 1 cm thick slab of silicone overlaying a calcium carbonate specimen and demonstrated a high differentiation of the two Raman signals. We reconstructed the Raman spectra of the two layers, offering the potential for improved and quantitative material analysis. Using a bilayer phantom made of porcine muscle and calcium carbonate, we proved that our system can retrieve Raman peaks even in the presence of autofluorescence typical of biomedical tissues. Overall, our novel TD-DIRS setup proposes a cost-effective and high-performance approach for in-depth Raman spectroscopy in diffusive media.

Photosystem II monomeric antenna CP26 plays a key role in nonphotochemical quenching in Chlamydomonas.

Thermal dissipation of excess excitation energy, called nonphotochemical quenching (NPQ), is 1 of the main photoprotective mechanisms in oxygenic photosynthetic organisms. Here, we investigated the function of the monomeric photosystem II (PSII) antenna protein CP26 in photoprotection and light harvesting in Chlamydomonas reinhardtii, a model organism for green algae. We used CRISPR/Cas9 genome editing and complementation to generate cp26 knockout mutants (named k6#) that did not negatively affect CP29 accumulation, which differed from previous cp26 mutants, allowing us to compare mutants specifically deprived of CP26, CP29, or both. The absence of CP26 partially affected PSII activity, causing reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to the wild type (Wt). This phenotype was fully rescued by genetic complementation and complemented strains accumulating different levels of CP26, demonstrating that ∼50% of CP26 content, compared to the Wt, was sufficient to restore the NPQ capacity. Our findings demonstrate a pivotal role for CP26 in NPQ induction, while CP29 is crucial for PSII activity. The genetic engineering of these 2 proteins could be a promising strategy to regulate the photosynthetic efficiency of microalgae under different light regimes.

Insight on the Intracellular Supramolecular Assembly of DTTO: A Peculiar Example of Cell-Driven Polymorphism.

The assembly of supramolecular structures within living systems is an innovative approach for introducing artificial constructs and developing biomaterials capable of influencing and/or regulating the biological responses of living organisms. By integrating chemical, photophysical, morphological, and structural characterizations, it is shown that the cell-driven assembly of 2,6-diphenyl-3,5-dimethyl-dithieno[3,2-b:2',3'-d]thiophene-4,4-dioxide (DTTO) molecules into fibers results in the formation of a "biologically assisted" polymorphic form, hence the term bio-polymorph. Indeed, X-ray diffraction reveals that cell-grown DTTO fibers present a unique molecular packing leading to specific morphological, optical, and electrical properties. Monitoring the process of fiber formation in cells with time-resolved photoluminescence, it is established that cellular machinery is necessary for fiber production and a non-classical nucleation mechanism for their growth is postulated. These biomaterials may have disruptive applications in the stimulation and sense of living cells, but more crucially, the study of their genesis and properties broadens the understanding of life beyond the native components of cells.

Membrane Order Effect on the Photoresponse of an Organic Transducer.

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation.

Role of Efficient Charge Transfer at the Interface between Mixed-Phase Copper-Cuprous Oxide and Conducting Polymer Nanostructures for Photocatalytic Water Splitting.

Photocatalytic hydrogen generation from water splitting is regarded as a sustainable technology capable of producing green solar fuels. However, the low charge separation efficiencies and the requirement of lowering redox potentials are unresolved challenges. Herein, a multiphase copper-cuprous oxide/polypyrrole (PPy) heterostructure has been designed to identify the role of multiple oxidation states of metal oxides in water reduction and oxidation. The presence of a mixed phase in PPy heterostructures enabled an exceptionally high photocatalytic H2 generation rate of 41 mmol h-1 with an apparent quantum efficiency of 7.2% under visible light irradiation, which is a 7-fold augmentation in contrast to the pure polymer. Interestingly, the copper-cuprous oxide/PPy heterostructures exhibited higher charge carrier density, low resistivity, and 6 times higher photocurrent density compared to Cu2O/PPy. Formation of a p-p-n junction between polymer and mixed-phase metal oxide interfaces induce a built-in electric field which influences directional charge transfer that improves the catalytic activity. Notably, photoexcited charge separation and transfer have been significantly improved between copper-cuprous oxide nanocubes and PPy nanofibers, as revealed by femtosecond transient absorption spectroscopy. Additionally, the photocatalyst demonstrates excellent stability without loss of catalytic activity during cycling tests. The present study highlights a superior strategy to boost photocatalytic redox reactions using a mixed-phase metal oxide in the heterostructure to achieve enhanced light absorption, longer charge carrier lifetimes, and highly efficient photocatalytic H2 and O2 generation.

Core-Shell Architecture in Poly(3-hexylthiophene) Nanoparticles: Tuning of the Photophysical Properties for Enhanced Neuronal Photostimulation.

This study shows that entirely thiophene-based core@shell nanoparticles, in which the shell is made of the oxidized form of the core polymer (P3HT@PTDOx NPs), result in a type II interface at the particle surface. This enables the development of advanced photon nanotransducers with unique chemical-physical and biofunctional properties due to the core@shell nanoarchitecture. We demonstrate that P3HT@PTDOx NPs present a different spatial localization of the excitation energy with respect to the nonoxidized NPs, showing a prevalence of surface states as a result of a different alignment of the HOMO/LUMO energy levels between the core and shell. This allows for the efficient photostimulation of retinal neurons. Indeed, thanks to the stronger and longer-lived charge separation, P3HT@PTDOx NPs, administered subretinally in degenerate retinas from the blind Royal College of Surgeons rats, are more effective in photostimulation of inner retinal neurons than the gold standard P3HT NPs.

Molecular mechanisms of light harvesting in the minor antenna CP29 in near-native membrane lipidic environment.

CP29, a chlorophyll a/b-xanthophyll binding protein, bridges energy transfer between the major LHCII antenna complexes and photosystem II reaction centers. It hosts one of the two identified quenching sites, making it crucial for regulated photoprotection mechanisms. Until now, the photophysics of CP29 has been 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 the Chl lifetime, which underlines the critical role of zeaxanthin in modulating photoprotection activity.

All-Polymer Microcavities for the Fluorescence Radiative Rate Modification of a Diketopyrrolopyrrole Derivative.

Controlling the radiative rate of emitters with macromolecular photonic structures promises flexible devices with enhanced performances that are easy to scale up. For instance, radiative rate enhancement empowers low-threshold lasers, while rate suppression affects recombination in photovoltaic and photochemical processes. However, claims of the Purcell effect with polymer structures are controversial, as the low dielectric contrast typical of suitable polymers is commonly not enough to provide the necessary confinement. Here we show all-polymer planar microcavities with photonic band gaps tuned to the photoluminescence of a diketopyrrolopyrrole derivative, which allows a change in the fluorescence lifetime. Radiative and nonradiative rates were disentangled systematically by measuring the external quantum efficiencies and comparing the planar microcavities with a series of references designed to exclude any extrinsic effects. For the first time, this analysis shows unambiguously the dye radiative emission rate variations obtained with macromolecular dielectric mirrors. When different waveguides, chemical environments, and effective refractive index effects in the structure were accounted for, the change in the radiative lifetime was assigned to the Purcell effect. This was possible through the exploitation of photonic structures made of polyvinylcarbazole as a high-index material and the perfluorinated Aquivion as a low-index one, which produced the largest dielectric contrast ever obtained in planar polymer cavities. This characteristic induces the high confinement of the radiation electric field within the cavity layer, causing a record intensity enhancement and steering the radiative rate. Current limits and requirements to achieve the full control of radiative rates with polymer planar microcavities are also addressed.

Azobenzene photoisomerization probes cell membrane viscosity.

The viscosity of cell membranes is a crucial parameter that affects the diffusion of small molecules both across and within the lipid membrane and that is related to several diseases. Therefore, the possibility to measure quantitatively membrane viscosity on the nanoscale is of great interest. Here, we report a complete investigation of the photophysics of an amphiphilic membrane-targeted azobenzene (ZIAPIN2) and we propose its use as a viscosity probe for cell membranes. We exploit ZIAPIN2 trans-cis photoisomerization to develop a molecular viscometer and to assess the viscosity of Escherichia coli bacteria membranes employing time-resolved fluorescence spectroscopy. Fluorescence lifetime measurements of ZIAPIN2 in E. coli bacteria suspensions correctly indicate that the membrane viscosity decreases as the temperature of the sample increases. Given the non-homogeneity and the anisotropy of cell membranes, as supported by the photophysical characterization of the probe within the lipid bilayer, we shed new light on the intricate membrane rheology.

Dynamics of Two Distinct Exciton Populations in Methyl-Functionalized Germanane.

Methyl-substituted germanane is an emerging material that has been proposed for novel applications in optoelectronics, photoelectrocatalysis, and biosensors. It is a two-dimensional semiconductor with a strong above-gap fluorescence associated with water intercalation. Here, we use time-resolved photoluminescence spectroscopy to understand the mechanism causing this fluorescence. We show that it originates from two distinct exciton populations. Both populations recombine exponentially, accompanied by the thermally activated transfer of exciton population from the shorter- to the longer-lived type. The two exciton populations involve different electronic levels and couple to different phonons. The longer-lived type of exciton migrates within the disordered energy landscape of localized recombination centers. These outcomes shed light on the fundamental optical and electronic properties of functionalized germanane, enabling the groundwork for future applications in optoelectronics, light harvesting, and sensing.

Compressed sensing in fluorescence microscopy.

Compressed sensing (CS) is a signal processing approach that solves ill-posed inverse problems, from under-sampled data with respect to the Nyquist criterium. CS exploits sparsity constraints based on the knowledge of prior information, relative to the structure of the object in the spatial or other domains. It is commonly used in image and video compression as well as in scientific and medical applications, including computed tomography and magnetic resonance imaging. In the field of fluorescence microscopy, it has been demonstrated to be valuable for fast and high-resolution imaging, from single-molecule localization, super-resolution to light-sheet microscopy. Furthermore, CS has found remarkable applications in the field of mesoscopic imaging, facilitating the study of small animals' organs and entire organisms. This review article illustrates the working principles of CS, its implementations in optical imaging and discusses several relevant uses of CS in the field of fluorescence imaging from super-resolution microscopy to mesoscopy.

Above pile-up fluorescence microscopy with a 32 Mc/s single-channel time-resolved SPAD system.

One of the major drawbacks of time-correlated single-photon counting (TCSPC) is generally represented by pile-up distortion, which strongly bounds the maximum acquisition speed to a few percent of the laser excitation rate. Based on a previous theoretical analysis, recently we presented the first, to the best of our knowledge, low-distortion and high-speed TCSPC system capable of overcoming the pile-up limitation by perfectly matching the single-photon avalanche diode (SPAD) dead time to the laser period. In this work, we validate the proposed system in a standard fluorescence measurement by comparing experimental data with the reference theoretical framework. As a result, a count rate of 32 Mc/s was achieved with a single-channel system still observing a negligible lifetime distortion.

Shedding Light on Thermally Induced Optocapacitance at the Organic Biointerface.

Photothermal perturbation of the cell membrane is typically achieved using transducers that convert light into thermal energy, eventually heating the cell membrane. In turn, this leads to the modulation of the membrane electrical capacitance that is assigned to a geometrical modification of the membrane structure. However, the nature of such a change is not understood. In this work, we employ an all-optical spectroscopic approach, based on the use of fluorescent probes, to monitor the membrane polarity, viscosity, and order directly in living cells under thermal excitation transduced by a photoexcited polymer film. We report two major results. First, we show that rising temperature does not just change the geometry of the membrane but indeed it affects the membrane dielectric characteristics by water penetration. Second, we find an additional effect, which is peculiar for the photoexcited semiconducting polymer film, that contributes to the system perturbation and that we tentatively assigned to the photoinduced polarization of the polymer interface.

The Role of Acidic Residues in the C Terminal Tail of the LHCSR3 Protein of Chlamydomonas reinhardtii in Non-Photochemical Quenching.

Light-harvesting complex stress-related (LHCSR) proteins in green algae are essential for photoprotection via a non-photochemical quenching (NPQ), playing the dual roles of pH sensing and dissipation of chlorophylls excited-state energy. pH sensing occurs via a protonation of acidic residues located mainly on its lumen-exposed C-terminus. Here, we combine in vivo and in vitro studies to ascertain the role in NPQ of these protonatable C-terminal residues in LHCSR3 from Chlamydomonas reinhardtii. In vivo studies show that four of the residues, D239, D240, E242, and D244, are not involved in NPQ. In vitro experiments on an LHCSR3 chimeric protein, obtained by a substitution of the C terminal with that of another LHC protein lacking acidic residues, show a reduction of NPQ compared to the wild type but preserve the quenching mechanism involving a charge transfer from carotenoids to chlorophylls. NPQ in LHCSR3 is thus a complex mechanism, composed of multiple contributions triggered by different acidic residues.