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The infrared beamline at BESSYâ 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 30â nm and infrared spectra to be collected from an effective volume of less than 30â nm × 30â nm × 12â nm. Potential steps for further optimization of the beamline performance are discussed.
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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.
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Pared Celular , Celulosa , Celulosa/química , Pared Celular/química , Membrana Celular , Diagnóstico por Imagen , AnisotropíaRESUMEN
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.
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Fiber-reinforced polymer composites are largely employed for their improved strength with respect to unfilled matrices. Considering semi-crystalline materials under relevant processing conditions, the applied pressure and flow induce shear stresses at the fiber-polymer interface. These stresses may strongly enhance the nucleation ability of the fiber surface with respect to the quiescent case. It is thus possible to assume that the fiber features are no longer of importance and that crystallization is dominated by the effect of flow. However, by making use of an advanced experimental technique, i.e., polarization-modulated synchrotron infrared microspectroscopy (PM-SIRMS), we are able to show that the opposite is true for the industrially relevant case of isotactic polypropylene (iPP). With PM-SIRMS, the local chain orientation is measured with micron-size spatial resolution. This orientation can be related to the polymer nucleation density along the fiber surface. For various combinations of an iPP matrix and fiber, the degree of orientation in the cylindrical layer that develops during flow correlates well with the differences in nucleation density found in quiescent conditions. This result shows that the morphological development during processing of polymer composites is not solely determined by the flow field, nor by the nucleating ability of the fiber surface alone, but rather by a synergistic combination of the two. In addition, using finite element modeling, it is demonstrated that, under the experimentally applied flow conditions, the interphase structure formation is mostly dominated by the rheological characteristics of the material rather than perturbations in experimental conditions, such as shear rate, layer thickness, and temperature. This once again highlights the importance of matrix-filler interplay during flow and, thus, of material selection in the design of hybrid and lightweight composite technologies.
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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.
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Petróleo , Hidrocarburos Policíclicos Aromáticos , Contaminantes Químicos del Agua , Biopelículas , Butadienos/análisis , Ecosistema , Monitoreo del Ambiente , Microplásticos , Nylons , Petróleo/análisis , Plásticos/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Polietileno/análisis , Tereftalatos Polietilenos , Polipropilenos/análisis , Poliestirenos/análisis , Cloruro de Polivinilo/análisis , Contaminantes Químicos del Agua/análisisRESUMEN
There has been extensive research in the field of material-extrusion (Mat-Ex) 3D printing to improve the inter-layer bonding process. Much research focusses on how various printing conditions may be detrimental to weld strength; many different feedstocks have been investigated along with various additives to improve strength. Surprisingly, there has been little attention directed toward how fundamental molecular properties of the feedstock, in particular the average molar mass of the polymer, may contribute to microstructure of the weld. Here we showed that weld strength increases with decreasing average molar mass, contrary to common observations in specimens processed in more traditional ways, e.g., by compression molding. Using a combination of synchrotron infra-red polarisation modulation microspectroscopy measurements and continuum modelling, we demonstrated how residual molecular anisotropy in the weld region leads to poor strength and how it can be eradicated by decreasing the relaxation time of the polymer. This is achieved more effectively by reducing the molar mass than by the usual approach of attempting to govern the temperature in this hard to control non-isothermal process. Thus, we propose that molar mass of the polymer feedstock should be considered as a key control parameter for achieving high weld strength in Mat-Ex.
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N-acylated substitutedß3oligoamides are known to form unique supramolecular nanorods based on a 3-point hydrogen bond self-assembly motif. This motif is an intermolecular extension of the hydrogen bonding network that stabilizes the 14-helix secondary structure unique toß3oligoamides. Acetylation of the N-terminus of the molecule provides the necessary third hydrogen bond pair of the motif. Here, the possibility of introducing the third hydrogen bond pair via amidation of the C terminus is investigated. While similar in purpose, this modification introduces a chemically distinct new self-assembly motif, also removing the bulky carboxyl group that does not fold into the 14 helix positioning instead as a side chain. Three substitutedß3oligoamide variants with the base sequence LIA (where the letters denoteß3residues with side chains analogous to α amino acids) were compared: N-acylated Ac-ß3[LIA] as a reference, C-amidatedß3[LIA]-CONH2, andß3[LIA] with free unmodified N and C termini as a negative control. The three variants were dissolved in water to promote self-assembly. The self-assembly was characterised using mid- and far-infrared spectroscopy, small angle x-ray scattering (SAXS) and atomic force microscopy (AFM). IR measurements confirmed that all three samples were in a similar conformation, consistent with pseudo 14-helical secondary structures. Far-infrared spectroscopy measurements ofß3[LIA]-CONH2showed distinct peaks consistent with highly organised skeletal modes, i.e. regular supramolecular assembly, that was largely absent from the other two oligoamides. Modelling of SAXS data is consistent with elliptical cylinder structures resulting from nanorod bundling for bothß3[LIA]-CONH2and Ac-ß3[LIA], but not in the unmodified sample. Consistently, AFM imaging showed large nanorod bundling structures inß3[LIA]-CONH2, varied bundling structures in Ac-ß3[LIA], and only aggregation inß3[LIA]. Amidation showed much more organised and robust assembly compared to acetylation, providing a new, easy to synthesize self-assembly motif for helical nanorod assembly that is similar but distinct to N-acylation.
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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 â¼ 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.
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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.
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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.
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Rodopsina/química , Imagen Individual de Molécula/instrumentación , Imagen Individual de Molécula/métodos , Espectrofotometría Infrarroja/instrumentación , Espectrofotometría Infrarroja/métodos , Cinética , Luz , Procesos Fotoquímicos , Conformación ProteicaRESUMEN
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.
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Bacteriorodopsinas/ultraestructura , Proteínas de la Membrana/ultraestructura , Proteínas Mutantes/ultraestructura , Bombas de Protones/ultraestructura , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Campos Electromagnéticos , Transporte Iónico/genética , Proteínas de la Membrana/química , Microscopía de Fuerza Atómica , Proteínas Mutantes/química , Proteínas Mutantes/genética , Nanotecnología/métodos , Conformación Proteica , Bombas de Protones/química , Membrana Púrpura/química , Membrana Púrpura/ultraestructura , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
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 â Bbmm transition, whereas the pressure-induced Pnma â Bbmm transformation at ambient temperature, accompanied by an electronic semiconductor â 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.
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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 â 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.
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Micelas , Polímeros/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
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.
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Proteínas de la Membrana/química , Nanotecnología/métodos , Membrana Púrpura/química , Imagenología Tridimensional , Conformación Proteica , Espectrofotometría InfrarrojaRESUMEN
Among optogenetic tools, channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.
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Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils.
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Amiloide/química , Cristalinas/química , Cristalino/metabolismo , Nanofibras/química , Nanopartículas/química , Animales , Benzotiazoles , Línea Celular , Supervivencia Celular , Neoplasias Endometriales/patología , Femenino , Peces , Humanos , Concentración de Iones de Hidrógeno , Insulina/química , Microscopía Electrónica de Transmisión , Péptido Hidrolasas/química , Solventes/química , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Tiazoles/química , Proteína de Suero de Leche/metabolismoRESUMEN
Infrared (IR) microspectroscopy has the capacity to determine the extent of phase separation in polymer blends. However, a major limitation in the use of this technique has been its reliance on overlapping peaks in the IR spectra to differentiate between polymers of similar chemical compositions in blends. The objective of this study was to evaluate the suitability of deuteration of one mixture component to separate infrared (IR) absorption bands and provide image contrast in phase separated materials. Deuteration of poly(3-hydroxyoctanoate) (PHO) was achieved via microbial biosynthesis using deuterated substrates, and the characteristic C-D stretching vibrations provided distinct signals completely separated from the C-H signals of protonated poly(3-hydroxybutyrate) (PHB). Phase separation was observed in 50:50 (% w/w) blends as domains up to 100 µm through the film cross sections, consistent with earlier reports of phase separation observed by scanning electron microscopy (SEM) of freeze-fractured protonated polymer blends. The presence of deuterated phases throughout the film suggests there is some miscibility at smaller length scales, which increased with increasing PHB content. These investigations indicate that biodeuteration combined with IR microspectroscopy represents a useful tool for mapping the phase behavior of polymer blends.
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Polímeros/química , Espectroscopía de Resonancia Magnética , Conformación Molecular , Espectrofotometría InfrarrojaRESUMEN
The application of FTIR spectroscopy to disease diagnosis requires a thorough knowledge of the spectroscopy associated with the cell cycle to discern disease markers from normal cellular events. We have applied synchrotron FTIR spectroscopy to monitor cells at different phases of the cell cycle namely G1, S and G2 phases. By applying Principal component analysis (PCA) from three independent trials we show clustering on a 2-dimensional scores plots (PC1 versus PC2) from cell spectra only two hours apart within the cell cycle. The corresponding PCA Loadings Plots indicate the clustering is primarily based on changes to the overall concentration of nucleic acids, proteins and lipids. During the first ten hours post mitosis, cells are observed to increase in protein and decrease in both lipid and nucleic acid concentration. During the synthesis phase, (beginning 9-11 hours post-mitosis) the PCA Loadings Plots show the accumulation of lipids within the cell as well the duplication of the genome as evidenced by strong DNA contributions. In the 4-6 hours following the synthesis phase, the cells once again accumulate protein while the relative nucleic acid and lipid concentrations decrease. These results, in comparison to previous studies on dehydrated cells, show previously unresolvable biochemical information as well as highlighting the advantages of FTIR spectroscopy applied to single living cells.
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Ciclo Celular/fisiología , Fenómenos Fisiológicos Celulares , Fibroblastos/citología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Sincrotrones , Animales , Células Cultivadas , Ratones , Análisis de Componente PrincipalRESUMEN
The wings of some insects, such as cicadae, have been reported to possess a number of interesting and unusual qualities such as superhydrophobicity, anisotropic wetting and antibacterial properties. Here, the chemical composition of the wings of the Clanger cicada (Psaltoda claripennis) were characterized using infrared (IR) microspectroscopy. In addition, the data generated from two separate synchrotron IR facilities, the Australian Synchrotron Infrared Microspectroscopy beamline (AS-IRM) and the Synchrotron Radiation Center (SRC), University of Wisconsin-Madison, IRENI beamline, were analysed and compared. Characteristic peaks in the IR spectra of the wings were assigned primarily to aliphatic hydrocarbon and amide functionalities, which were considered to be an indication of the presence of waxy and proteinaceous components, respectively, in good agreement with the literature. Chemical distribution maps showed that, while the protein component was homogeneously distributed, a significant degree of heterogeneity was observed in the distribution of the waxy component, which may contribute to the self-cleaning and aerodynamic properties of the cicada wing. When comparing the data generated from the two beamlines, it was determined that the SRC IRENI beamline was capable of producing higher-spatial-resolution distribution images in a shorter time than was achievable at the AS-IRM beamline, but that spectral noise levels per pixel were considerably lower on the AS-IRM beamline, resulting in more favourable data where the detection of weak absorbances is required. The data generated by the two complementary synchrotron IR methods on the chemical composition of cicada wings will be immensely useful in understanding their unusual properties with a view to reproducing their characteristics in, for example, industry applications.
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Hemípteros/química , Espectrofotometría Infrarroja/métodos , Sincrotrones , Termografía/métodos , Alas de Animales/química , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
Organic carbon is a critical component of aquatic systems, providing energy storage and transfer between organisms. Fungi are a major decomposer group in the aquatic carbon cycle, and are one of few groups thought to be capable of breaking down woody (lignified) tissue. In this work we have used high spatial resolution (synchrotron light source) infrared micro-spectroscopy to study the interaction between aquatic fungi and lignified leaf vein material (xylem) from River Redgum trees (E. camaldulensis) endemic to the lowland rivers of South-Eastern Australia. The work provides spatially explicit evidence that fungal colonisation of leaf litter involves the oxidative breakdown of lignin immediately adjacent to the fungal tissue and depletion of the lignin-bound cellulose. Cellulose depletion occurs over relatively short length scales (5-15 µm) and highlights the likely importance of mechanical breakdown in accessing the carbohydrate content of this resource. Low bioavailability compounds (oxidized lignin and polyphenols of plant origin) remain in colonised leaves, even after fungal activity diminishes, and suggests a possible pathway for the sequestration of carbon in wetlands. The work shows that fungi likely have a critical role in the partitioning of lignified material into a biodegradable fraction that can re-enter the aquatic carbon cycle, and a recalcitrant fraction that enters long-term storage in sediments or contribute to the formation of dissolved organic carbon in the water column.