Infrared spectroscopy across scales in length and time at BESSY†II.

The infrared beamline at BESSY II storage ring was upgraded recently to extend the capabilities of infrared microscopy. The endstations available at the beamline are now facilitating improved characterization of molecules and materials at different length scales and time resolutions. Here, the current outline of the beamline is reported and an overview of the endstations available is given. In particular, the first results obtained by using a new microscope for nano-spectroscopy that was implemented are presented. The capabilities of the scattering-type near-field optical microscope (s-SNOM) are demonstrated by investigating cellulose microfibrils, representing nanoscopic objects of a hierarchical structure. It is shown that the s-SNOM coupled to the beamline allows imaging to be performed with a spatial resolution of less than 30 nm and infrared spectra to be collected from an effective volume of less than 30 nm × 30 nm × 12 nm. Potential steps for further optimization of the beamline performance are discussed.

In situ infrared imaging of the local orientation of cellulose fibrils in plant secondary cell walls.

The mechanical and chemical properties of plant cell walls greatly rely on the supramolecular assembly of cellulose fibrils. To study the local orientation of cellulose in secondary plant cell walls, diffraction limited infrared (IR) micro-spectroscopic mapping experiments were conducted at different orientation of transverse leaf section of the grass Sorghum bicolor with respect to the polarization direction of the IR radiation. Two-dimensional maps, based on polarization-sensitive absorption bands of cellulose were obtained for different polarization angles. They reveal a significant degree of anisotropy of the cellulose macromolecules as well as of other biopolymers in sclerenchyma and xylem regions of the cross section. Quantification of the signals assigned to polarization sensitive vibrational modes allowed to determine the preferential orientation of the sub-micron cellulose fibrils in single cell walls. A sample of crystalline nano-cellulose comprising both a single microcrystal as well as unordered layers of nanocrystals was used for validation of the approach. The results demonstrate that diffraction limited IR micro-spectroscopy can be used to study hierarchically structured materials with complex anisotropic behavior.

SAXS signature of the lamellar ordering of ionic domains of perfluorinated sulfonic-acid ionomers by electric and magnetic field-assisted casting.

At present, small angle X-ray scattering (SAXS) studies of perfluorinated sulfonic-acid ionomers (PFSAs) are unable to fully determine the true shape of their building blocks, as recent SAXS modelling predicts disk- and rod-like nanoionic domains as being equally possible. This scenario requires evidence-based findings to unravel the real shape of PFSA building blocks. Herein, a SAXS pattern signature for a lamellar nanophase separation of the ionic domains of Nafion is presented, backed by mid and far infrared spectroscopy (MIR and FIR) and wide angle X-ray scattering (WAXS) data of Nafion in different ionic forms, a broad range of ionic phase contents (EW ∼ 859-42 252 g eq-1) and temperatures. The study indicates that the lamellar arrangement of the ionic domains is the most representative morphology that accounts for the physical properties of this ionomer. The lamellar SAXS reflections of Nafion are enhanced in electric and magnetic field-aligned membranes, as confirmed by atomic force microscopy (AFM). Electric and magnetic field-assisted casting of Nafion allowed producing nanostructured and anisotropic films with the lamellas stacked perpendicularly to the field vector, which is the direction of interest for several applications. Such nanostructured Nafion membranes are bestowed with advanced optical and proton transport properties, making them promising materials for solar and fuel cells.

Polarizing interferometer for the unambiguous determination of the ellipsometric parameters.

We report on a polarizing interferometer-ellipsometer arrangement that overcomes the need for additional measurements with a retarder for the unambiguous determination of the ellipsometric parameters in the far infrared spectral range. It consists of a Martin-Puplett interferometer and a wire-grid polarizer as an analyzer. The application of such interferometer-ellipsometer is experimentally demonstrated on a polyethylene sample deriving the refractive index and the thickness in the spectral range between 15 and 35cm-1. Based on these results, a similar solution without a retarder for the mid-infrared spectral region is additionally proposed.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450-1800 cm-1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Effects of temperature and pressure on the optical and vibrational properties of thermoelectric SnSe.

We have conducted a comprehensive investigation of the optical and vibrational properties of the binary semiconductor SnSe as a function of temperature and pressure by means of experimental and ab initio probes. Our high-temperature investigations at ambient pressure have successfully reproduced the progressive enhancement of the free carrier concentration upon approaching the Pnma → Bbmm transition, whereas the pressure-induced Pnma → Bbmm transformation at ambient temperature, accompanied by an electronic semiconductor → semi-metal transition, has been identified for bulk SnSe close to 10 GPa. Modeling of the Raman-active vibrations revealed that three-phonon anharmonic processes dominate the temperature-induced mode frequency evolution. In addition, SnSe was found to exhibit a pressure-induced enhancement of the Born effective charge. Such behavior is quite unique and cannot be rationalized within the proposed effective charge trends of binary materials under pressure.

Heterogeneity of the Transmembrane Protein Conformation in Purple Membranes Identified by Infrared Nanospectroscopy.

Cell membranes are intrinsically heterogeneous, as the local protein and lipid distribution is critical to physiological processes. Even in template systems embedding a single protein type, like purple membranes, there can be a different local response to external stimuli or environmental factors, resulting in heterogeneous conformational changes. Despite the dramatic advances of microspectroscopy techniques, the identification of the conformation heterogeneity is still a challenging task. Tip-enhanced infrared nanospectroscopy is here used to identify conformational changes connected to the hydration state of the transmembrane proteins contained in a 50 nm diameter cell membrane area, without the need for fluorescent labels. In dried purple membrane monolayers, areas with fully hydrated proteins are found among large numbers of molecules with randomly distributed hydration states. Infrared nanospectroscopy results are compared to the spectra obtained with diffraction-limited infrared techniques based on the use of synchrotron radiation, in which the diffraction limit still prevents the observation of nanoscale heterogeneity.

Anisotropy in bone demineralization revealed by polarized far-IR spectroscopy.

Bone material is composed of an organic matrix of collagen fibers and apatite nanoparticles. Previously, vibrational spectroscopy techniques such as infrared (IR) and Raman spectroscopy have proved to be particularly useful for characterizing the two constituent organic and inorganic phases of bone. In this work, we tested the potential use of high intensity synchrotron-based far-IR radiation (50-500 cm(-1)) to gain new insights into structure and chemical composition of bovine fibrolamellar bone. The results from our study can be summarized in the following four points: (I) compared to far-IR spectra obtained from synthetic hydroxyapatite powder, those from fibrolamellar bone showed similar peak positions, but very different peak widths; (II) during stepwise demineralization of the bone samples, there was no significant change neither to far-IR peak width nor position, demonstrating that mineral dissolution occurred in a uniform manner; (III) application of external loading on fully demineralized bone had no significant effect on the obtained spectra, while dehydration of samples resulted in clear differences. (IV) using linear dichroism, we showed that the anisotropic structure of fibrolamellar bone is also reflected in anisotropic far-IR absorbance properties of both the organic and inorganic phases. Far-IR spectroscopy thus provides a novel way to functionally characterize bone structure and chemistry, and with further technological improvements, has the potential to become a useful clinical diagnostic tool to better assess quality of collagen-based tissues.

Vibrational signature of hydrated protons confined in MXene interlayers.

The hydration structure of protons has been studied for decades in bulk water and protonated clusters due to its importance but has remained elusive in planar confined environments. Two-dimensional (2D) transition metal carbides known as MXenes show extreme capacitance in protic electrolytes, which has attracted attention in the energy storage field. We report here that discrete vibrational modes related to protons intercalated in the 2D slits between Ti3C2Tx MXene layers can be detected using operando infrared spectroscopy. The origin of these modes, not observed for protons in bulk water, is attributed to protons with reduced coordination number in confinement based on Density Functional Theory calculations. This study therefore demonstrates a useful tool for the characterization of chemical species under 2D confinement.

IR and X-ray time-resolved simultaneous experiments: an opportunity to investigate the dynamics of complex systems and non-equilibrium phenomena using third-generation synchrotron radiation sources.

Third-generation storage rings are modern facilities working with high currents and designed to host powerful radiation sources, like undulators and wigglers, and to deliver high-brilliance beams to users. Many experiments at high spatial resolution, such as spectromicroscopy at the nanometre scale and with high temporal resolution to investigate kinetics down to the picosecond regime, are now possible. The next frontier is certainly the combination of different methods in a unique set-up with the ultimate available spatial and temporal resolutions. In the last decade much synchrotron-based research has exploited the advantage of complementary information provided by time-resolved X-ray techniques and optical methods in the UV/Vis and IR domains. New time-resolved and concurrent approaches are necessary to characterize complex systems where physical-chemical phenomena occur under the same experimental conditions, for example to detect kinetic intermediates via complementary but independent observations. In this contribution we present scientific cases from original works and literature reviews to support the proposed IR/X-ray simultaneous approach, with both probes exploiting synchrotron radiation sources. In addition, simple experimental layouts that may take advantage of the high brilliance and the wide spectral distribution of the synchrotron radiation emission will be given for specific researches or applications to investigate dynamic processes and non-equilibrium phenomena occurring in many condensed matter and biological systems, of great interest for both fundamental research and technological applications.

Occurrence of polycyclic aromatic hydrocarbons, microplastics and biofilms in Alqueva surface water at touristic spots.

Freshwater pollution is a huge concern. A study aiming to evaluate physico-chemical characteristics, microbiota, occurrence of two groups of persistent environmental pollutants with similar chemical properties (polycyclic aromatic hydrocarbons- PAHs and microplastics - MPs) in Alqueva's surface water was performed during 2021. Water samples were collected at three spots related to touristic activities (two beaches and one marina) during the Winter, Spring, Summer and Autumn seasons. In addition, the presence of biofilms on plastic and natural materials (stone, wood/ vegetal materials) were assessed and compared. Water quality based on physicochemical parameters was acceptable with a low eutrophication level. PAHs concentration levels were lower than the standard limits established for surface waters by international organizations. However, carcinogenic compounds were detected in two sampling locations, which can pose a problem for aquatic ecosystems. PAHs profiles showed significant differences when comparing the dry seasons with the rainy seasons, with a higher number of different compounds detected in Spring. Low molecular weigh compounds, usually associated with the atmospheric deposition and petroleum contamination, were more prevalent. MPs were detected in all samples except one during the Winter season. The polymers detected were poly(methyl-2-methylpropenoate), polystyrene, polyethylene terephthalate, polyamide, polypropylene, styrene butadiene, polyvinyl chloride and low /high density polyethylene with the last being the most frequent. Biofilms were more often detected on plastics than on natural materials. In addition, biofilms detected on plastics were more complex with higher microbial diversity (e.g., bacteria, fungi/yeast and phytoplancton organisms) and richer in extrapolymeric material. Based on morphological analysis a good agreement between microbiota and microorganism present in the biofilms was found. Among microbiota were identified microorganisms previously linked to plastic and PAHs detoxification suggesting the need for further studies to evaluate the viability of using biofilms as part of a green bioremediation strategy to mitigate water pollution.

Shared solvation of sodium ions in alcohol-water solutions explains the non-ideality of free energy of solvation.

In order to explain the discrepancies between theories and experiments regarding the non-ideality in the free energy of solvation, here we present a microscopic picture of sodium ions dissolved in water-alcohol mixed solvents. We used X-ray absorption spectroscopy to probe the K-edge of sodium ions in mixed solvents of water and alcohols (methanol, ethanol) and in the respective pure solvents. In the mixed solvents a shared solvation of the sodium ions is observed. We find that specifically the water component plays a key role in stabilizing the solvation shell in mixed solvents, which was revealed by a selective photochemical process occurring only in the pure alcohol solvents.

Infrared and X-ray simultaneous spectroscopy: a novel conceptual beamline design for time resolved experiments.

Many physical/chemical processes such as metal-insulator transitions or self-assembly phenomena involve correlated changes of electronic and atomic structure in a wide time range from microseconds to minutes. To investigate these dynamic processes we not only need a highly brilliant photon source in order to achieve high spatial and time resolution but new experimental methods have to be implemented. Here we present a new optical layout for performing simultaneous or concurrent infrared and X-ray measurements. This approach may indeed return unique information for example the interplay between structural changes and chemical processes occurring in the investigated sample. A beamline combining two X-ray and IR beams may really take advantage of the unique synchrotron radiation properties: the high brilliance and the broad spectrum. In this contribution we will describe the conceptual layout and the expected performance of a complex system designed to collect IR and X-ray radiation from the same bending magnet on a third-generation synchrotron radiation ring. If realized, this beamline will enable time-resolved spectroscopy experiments offering new scientific opportunities at the frontiers of science.

Microscale imaging of the preservation state of 5,000-year-old archaeological bones by synchrotron infrared microspectroscopy.

Archaeological bone materials record characteristic markers of life in prehistoric times (dating, climate, environment, diet, human migration) in their isotopic and chemical composition in addition to palaeontological, archaeozoological, anthropological and palaeogenetic information. Thus, the discovery and conservation of archaeological bone materials is of great importance to get access to this information. However, archaeological materials are altered by different postmortem processes and it appears necessary to estimate if the archaeological information is still reliable or if it has been modified during burial. As archaeological bone materials present a high structural hierarchy at the micro- and nanoscale, changes induced by diagenetic phenomena have to be observed at these scales. One method for revealing post mortem changes of the bone structure and composition at the microscale is synchrotron radiation micro-FTIR imaging (SR micro-FTIR). Thus, thin sections of about 5,000-year-old archaeological bones have been analysed in transmission mode at the IRIS beamline (BESSY II, HZB Berlin) to determine markers of the state of bone preservation at the microscale. The archaeological bone material comes from station 19 of the Neolithic site of the Chalain Lake. By using SR micro-FTIR it was possible to image characteristic bone structures, e.g. osteons (the constitutive histological unit of cortical bone), using the absorption band ratios corresponding to different chemical bone constituents (collagen content and quality, phosphate crystallinity, carbonate content). These data allow us to precisely evaluate the state of preservation of a 5,000-year-old bone at the histological level.

Removal of Etalon Features in the Far-Infrared-Terahertz Transmittance Spectra of Thin Polymer Films.

Etalon features in infrared spectra of stratified samples, their influence on the interpretation, and methods to circumvent their presence in infrared spectra have been in discussion for decades. This paper focuses on the application of a method originally developed to remove interference fringes in the mid-infrared spectra for far-infrared Fourier transform spectroscopy on thin polymer films. We show that the total transmittance reflectance technique, commonly used for mid-infrared, also works successfully in the far-infrared spectral range where other approaches fail. Experimental spectra obtained by such technique are supported by model calculations and reveal the possibility and limits to obtain almost undisturbed far-infrared spectra which are suitable to determine low-energy vibrations of ionomer salts under certain sample conditions.

Application of terahertz spectroscopy to time-dependent chemical-physical phenomena.

We present here a "proof of concepts" experiment that has been realized to show that time-dependent phenomena can be successfully studied in the terahertz region in a non pump-probe configuration. We have built-up an original analytical setup that has allowed following the evaporation of a deuterated water droplet cast on a CVD diamond substrate simultaneously in the near-middle infrared region and in the terahertz range. We have used a synchrotron light source in the terahertz region and a conventional thermal source in the infrared range. The results demonstrate that it is possible to study time-dependent phenomena simultaneously in the middle and terahertz ranges monitoring the entire chemical-physical process that occurs in the time domain of minutes.

Féry Infrared Spectrometer for Single-Shot Analysis of Protein Dynamics.

Current submillisecond time-resolved broad-band infrared spectroscopy, one of the most frequently used techniques for studying structure-function relationships in life sciences, is typically limited to fast-cycling reactions that can be repeated thousands of times with high frequency. Notably, a majority of chemical and biological processes do not comply with this requirement. For example, the activation of vertebrate rhodopsin, a prototype of many protein receptors in biological organisms that mediate basic functions of life, including vision, smell, and taste, is irreversible. Here we present a dispersive single-shot Féry spectrometer setup that extends such spectroscopy to irreversible and slow-cycling systems by exploiting the unique properties of brilliant synchrotron infrared light combined with an advanced focal plane detector array embedded in a dispersive optical concept. We demonstrate our single-shot method on microbial actinorhodopsin with a slow photocycle and on vertebrate rhodopsin with irreversible activation.

Systemic ubiquitin release after blunt trauma and burns: association with injury severity, posttraumatic complications, and survival.

BACKGROUND: Recent data suggest that ubiquitin (Ub) is systemically released after trauma, has pleiotropic effects on host defense mechanisms, and that Ub administration reduces fluid shifts into tissues during inflammation. Ub release after burns (B) has not been studied and its association with injury severity and outcome after blunt trauma (T) is unknown. Thus, we evaluated Ubs association with injury severity and outcomes after B and T. METHODS: Injury severity was assessed with the Injury Severity Score (ISS) in T and burn size (% total body surface area, %TBSA) in B. A total of 129 T (ISS: 26 +/- 13) and 55 B (46% +/- 18% TBSA) were observed for sepsis/multiple organ failure (MOF) and survival. In B, sequential organ failure assessment scores were documented daily. Fifty volunteers served as controls (C) Ub serum levels were measured on day 0 (admission), 1, 3, 5, and 7 by enzyme-linked immunosorbent assay. Data were analyzed using bivariate or partial correlation analyses, t test, and analysis of variance with Tukey post-hoc test for multiple comparisons (two-tailed p < 0.05). RESULTS: Ub was significantly elevated in patients. Peak levels (ng/mL) were detectable on day 0 (C: 118 +/- 76; T: 359 +/- 205; B: 573 +/- 331) and increased with increased ISS, %TBSA, and presence of inhalation injury. In T, Ub normalized by day 3, but remained elevated in B. In B, Ub correlated significantly negative with sequential organ failure assessment scores (r: -0.143; p = 0.0147), sepsis/MOF development (r: -0.363; p = 0.001), and survival (r: -0.231; p = 0.009). Compared with B who recovered uneventfully, Ub levels were significantly lower on days 1 to 7 and on days 5/7 in B who developed sepsis/MOF or died, respectively. CONCLUSION: Ub concentrations reflect the extent of tissue damage. Along with Ubs previously described anti- inflammatory properties, this study suggests that its systemic release is protective, that burn patients who develop sepsis/MOF have a relative Ub deficiency and that Ub could play an important role during the physiologic response to burn injury.

Interplay of α/ß-Relaxation Dynamics and the Shape of Ionomer Building Blocks.

The relation between the α/ß relaxations and the shape of the building blocks of ionomer materials is a key factor for programming an important temperature-dependent property: the memory of shape. However, the morphology of ionomers is indirectly obtained via modeling of small angle X-ray scattering (SAXS) data owing to the hardly accessible image characterization of the nanometric building blocks ‒ micelle-like cylindrical polymeric aggregates (radius ~2-6 nm and length >100 nm). Herein, broadband dielectric spectroscopy (BDS) measurements, free from electrode polarization effects, allowed identifying the time and temperature dependence of the polarization of different length scales of the ionomer matrix, and more importantly, by directly providing the aspect ratio of the radius and length of the polymeric aggregates for each desired temperature. This finding is essential for controlling the shape of ionomer based functional products under several stimuli conditions, thereby advancing remarkable applications, such as four dimensional (4D) printing.

Characterization of temperature-dependent iron-imidazole vibrational modes in far infrared.

The active site of several oxygen binding proteins can be mimicked with the ferric iron protoporphyrin IX derivative hemin, coordinating two imidazole molecules and embedded in sodium dodecyl sulfate (SDS) micelles; the detergent simulates the hydrophobic cavity of heme proteins. We studied the low-frequency vibrational modes of the porphyrin-iron-imidazole bonding in infrared absorbance spectra. Assignment of the metal-ligand vibrations to signals at 396, 387, and 378 cm(-1) was performed by isotope labeling of the imidazole ligand. These modes were also found to be temperature-dependent and to display a linear increase of signal intensity between 25 and 150 K and, with a different slope, between 150 and 300 K. The modes at 396 and 399 cm(-1) show for 25 K an up-shift about 4 cm(-1) and the signal at 378 cm(-1) a small downshift, indicating the involvement of antisymmetric stretching modes and, in the latter, of bending motions. Anharmonic couplings to doming modes are discussed, and the doming mode and hydrogen-bonding signature spectral range between 300 and 100 cm(-1) is presented.