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.

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.

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.

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.

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.

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.

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.

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.

Multispectral compressive fluorescence lifetime imaging microscopy with a SPAD array detector.

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 18×1 single-photon avalanche diode array detector working in a single-pixel camera scheme, which parallelizes the spectral detection, reducing measurement time. The proposed system, which implements a single-pixel camera with a compressive sensing scheme, represents an optimal microscopy framework towards the design of λFLIM setups.

Evolutionary divergence of photoprotection in the green algal lineage: a plant-like violaxanthin de-epoxidase enzyme activates the xanthophyll cycle in the green alga Chlorella vulgaris modulating photoprotection.

The xanthophyll cycle is the metabolic process by which the carotenoid violaxanthin is de-epoxidated to zeaxanthin, a xanthophyll with a crucial photoprotective role in higher plants and mosses. The role of zeaxanthin is still unclear in green algae, and a peculiar violaxanthin de-epoxidating enzyme was found in the model organism Chlamydomonas reinhardtii. Here, we investigated the molecular details and functions of the xanthophyll cycle in the case of Chlorella vulgaris, one of the green algae most considered for industrial cultivation, where resistance to high light stress is a prerequisite for sustainable biomass production. Identification of the violaxanthin de-epoxidase enzyme in C. vulgaris was performed by genome mining and in vitro analysis of the catalytic activity of the gene product identified. The photoprotective role of zeaxanthin was then investigated in vivo and in isolated pigment-binding complexes. The results obtained demonstrate the functioning, even though with a different pH sensitivity, of a plant-like violaxanthin de-epoxidase enzyme in C. vulgaris. Differently from C. reinhardtii, zeaxanthin accumulation in C. vulgaris was found to be crucial for photoprotective quenching of excitation energy harvested by both photosystem I and II. These findings demonstrate an evolutionary divergence of photoprotective mechanisms among Chlorophyta.

The Photophysics of Polythiophene Nanoparticles for Biological Applications.

In this work the photophysics of poly(3-hexylthiophene) nanoparticles (NPs) is investigated in the context of their biological applications. The NPs, made as colloidal suspensions in aqueous buffers, present a distinct absorption band in the low-energy region. On the basis of systematic analysis of absorption and transient absorption (TA) spectra taken under different pH conditions, this band is associated with charge-transfer states generated by the polarization of loosely bound polymer chains and originating from complexes formed with electron-withdrawing species. Importantly, the ground-state depletion of these states upon photoexcitation is active even on microsecond timescales, thus suggesting that they act as precursor states for long-living polarons; this could be beneficial for cellular stimulation. Preliminary transient absorption microscopy results for NPs internalized within the cells reveal the presence of long-living species, further substantiating their relevance in biointerfaces.

Fluorescence lifetime imaging of intracellular magnesium content in live cells.

The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.

Time-resolved multispectral imaging based on an adaptive single-pixel camera.

Time-resolved multispectral imaging has many applications in different fields, which range from characterization of biological tissues to environmental monitoring. In particular, optical techniques, such as lidar and fluorescence lifetime imaging, require imaging at the subnanosecond scales over an extended area. In this paper, we demonstrate experimentally a time-resolved multispectral acquisition scheme based on single-pixel imaging. Single-pixel imaging is an emerging paradigm that provides low-cost high-quality images. Here, we use an adaptive strategy that allows acquisition and image reconstruction times to be reduced drastically or full basis scans. Adaptive time-resolved multispectral imaging scheme can have significant applications in biological imaging, at scales from macroscopic to microscopic.

Time- and frequency-resolved fluorescence with a single TCSPC detector via a Fourier-transform approach.

We introduce a broadband single-pixel spectro-temporal fluorescence detector, combining time-correlated single photon counting (TCSPC) with Fourier transform (FT) spectroscopy. A birefringent common-path interferometer (CPI) generates two time-delayed replicas of the sample's fluorescence. Via FT of their interference signal at the detector, we obtain a two-dimensional map of the fluorescence as a function of detection wavelength and emission time, with high temporal and spectral resolution. Our instrument is remarkably simple, as it only requires the addition of a CPI to a standard single-pixel TCSPC system, and it shows a readily adjustable spectral resolution with inherently broad bandwidth coverage.

Excitation-emission Fourier-transform spectroscopy based on a birefringent interferometer.

The correlation of molecular excitation and emission events provides a powerful multidimensional spectroscopy tool, by relating transitions from electronic ground and excited states through two-dimensional excitation-emission maps. Here we present a compact, fast and versatile Fourier-transform spectrometer, combining absorption and excitation-emission fluorescence spectroscopy in the visible. We generate phase-locked excitation pulse pairs via an inherently stable birefringent wedge-based common-path interferometer, retaining all the advantages of Fourier-transform spectroscopy but avoiding active stabilization or auxiliary tracking beams. We employ both coherent and incoherent excitation sources on dye molecules in solution, with data acquisition times in the range of seconds and minutes, respectively.

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii.

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.

Multiple-view diffuse optical tomography system based on time-domain compressive measurements.

Compressive sensing is a powerful tool to efficiently acquire and reconstruct an image even in diffuse optical tomography (DOT) applications. In this work, a time-resolved DOT system based on structured light illumination, compressive detection, and multiple view acquisition has been proposed and experimentally validated on a biological tissue-mimicking phantom. The experimental scheme is based on two digital micromirror devices for illumination and detection modulation, in combination with a time-resolved single element detector. We fully validated the method and demonstrated both the imaging and tomographic capabilities of the system, providing state-of-the-art reconstruction quality.

Regulation of photosystem I light harvesting by zeaxanthin.

In oxygenic photosynthetic eukaryotes, the hydroxylated carotenoid zeaxanthin is produced from preexisting violaxanthin upon exposure to excess light conditions. Zeaxanthin binding to components of the photosystem II (PSII) antenna system has been investigated thoroughly and shown to help in the dissipation of excess chlorophyll-excited states and scavenging of oxygen radicals. However, the functional consequences of the accumulation of the light-harvesting complex I (LHCI) proteins in the photosystem I (PSI) antenna have remained unclarified so far. In this work we investigated the effect of zeaxanthin binding on photoprotection of PSI-LHCI by comparing preparations isolated from wild-type Arabidopsis thaliana (i.e., with violaxanthin) and those isolated from the A. thaliana nonphotochemical quenching 2 mutant, in which violaxanthin is replaced by zeaxanthin. Time-resolved fluorescence measurements showed that zeaxanthin binding leads to a previously unrecognized quenching effect on PSI-LHCI fluorescence. The efficiency of energy transfer from the LHCI moiety of the complex to the PSI reaction center was down-regulated, and an enhanced PSI resistance to photoinhibition was observed both in vitro and in vivo. Thus, zeaxanthin was shown to be effective in inducing dissipative states in PSI, similar to its well-known effect on PSII. We propose that, upon acclimation to high light, PSI-LHCI changes its light-harvesting efficiency by a zeaxanthin-dependent quenching of the absorbed excitation energy, whereas in PSII the stoichiometry of LHC antenna proteins per reaction center is reduced directly.

Nondestructive optical detection of monomer uptake in wood polymer composites.

A noninvasive method to assess the local monomer concentration within a wooden matrix, post monomer impregnation, by time-resolved diffuse optical spectroscopy is demonstrated. A data analysis technique for improving accuracy, which takes account of changes in the refractive index during the monomer uptake, has been employed. This technique can be potentially applied in the wood industry for the study of polymer composites as well as in cultural heritage science for noninvasively monitoring the penetration of chemical compounds used for consolidation or conservation purposes.

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA.

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.