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Brain iron content is widely reported to increase during "ageing", across multiple species from nematodes, rodents (mice and rats) and humans. Given the redox-active properties of iron, there has been a large research focus on iron-mediated oxidative stress as a contributor to tissue damage during natural ageing, and also as a risk factor for neurodegenerative disease. Surprisingly, however, the majority of published studies have not investigated brain iron homeostasis during the biological time period of senescence, and thus knowledge of how brain homeostasis changes during this critical stage of life largely remains unknown. This commentary examines the literature published on the topic of brain iron homeostasis during ageing, providing a critique on limitations of currently used experimental designs. The commentary also aims to highlight that although much research attention has been given to iron accumulation or iron overload as a pathological feature of ageing, there is evidence to support functional iron deficiency may exist, and this should not be overlooked in studies of ageing or neurodegenerative disease.
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Envejecimiento , Encéfalo , Hierro , Envejecimiento/metabolismo , Hierro/metabolismo , Humanos , Animales , Encéfalo/metabolismo , HomeostasisRESUMEN
Several lines of study suggest that peripheral metabolism of amyloid beta (Aß) is associated with risk for Alzheimer disease (AD). In blood, greater than 90% of Aß is complexed as an apolipoprotein, raising the possibility of a lipoprotein-mediated axis for AD risk. In this study, we report that genetic modification of C57BL/6J mice engineered to synthesise human Aß only in liver (hepatocyte-specific human amyloid (HSHA) strain) has marked neurodegeneration concomitant with capillary dysfunction, parenchymal extravasation of lipoprotein-Aß, and neurovascular inflammation. Moreover, the HSHA mice showed impaired performance in the passive avoidance test, suggesting impairment in hippocampal-dependent learning. Transmission electron microscopy shows marked neurovascular disruption in HSHA mice. This study provides causal evidence of a lipoprotein-Aß /capillary axis for onset and progression of a neurodegenerative process.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides/biosíntesis , Hepatocitos/metabolismo , Péptidos beta-Amiloides/genética , Animales , Barrera Hematoencefálica/patología , Encéfalo/irrigación sanguínea , Capilares/patología , Modelos Animales de Enfermedad , Humanos , Inflamación , Aprendizaje , Lipoproteínas/metabolismo , Masculino , Ratones Transgénicos , Degeneración NerviosaRESUMEN
Neurobiological research relies heavily on imaging techniques, such as fluorescence microscopy, to understand neurological function and disease processes. However, the number and variety of fluorescent probes available for ex vivo tissue section imaging limits the advance of research in the field. In this review, we outline the current range of fluorescent probes that are available to researchers for ex vivo brain section imaging, including their physical and chemical characteristics, staining targets, and examples of discoveries for which they have been used. This review is organised into sections based on the biological target of the probe, including subcellular organelles, chemical species (e.g., labile metal ions), and pathological phenomenon (e.g., degenerating cells, aggregated proteins). We hope to inspire further development in this field, given the considerable benefits to be gained by the greater availability of suitably sensitive probes that have specificity for important brain tissue targets.
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Pathogen attacks elicit dynamic and widespread molecular responses in plants. While our understanding of plant responses has advanced considerably, little is known of the molecular responses in the asymptomatic 'green' regions adjoining lesions. Here, we explore gene expression data and high-resolution elemental imaging to report the spatiotemporal changes in the asymptomatic green region of susceptible and moderately resistant wheat cultivars infected with a necrotrophic fungal pathogen, Pyrenophora tritici-repentis. We show, with improved spatiotemporal resolution, that calcium oscillations are modified in the susceptible cultivar, resulting in 'frozen' host defence signals at the mature disease stage, and silencing of the host's recognition and defence mechanisms that would otherwise protect it from further attacks. In contrast, calcium accumulation and a heightened defence response were observed in the moderately resistant cultivar in the later stage of disease development. Furthermore, in the susceptible interaction, the asymptomatic green region was unable to recover after disease disruption. Our targeted sampling technique also enabled detection of eight previously predicted proteinaceous effectors in addition to the known ToxA effector. Collectively, our results highlight the benefits of spatially resolved molecular analysis and nutrient mapping to provide high-resolution spatiotemporal snapshots of host-pathogen interactions, paving the way for disentangling complex disease interactions in plants.
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Transcriptoma , Triticum , Triticum/genética , Triticum/microbiología , Rayos X , Susceptibilidad a Enfermedades , Microscopía Fluorescente , Enfermedades de las Plantas/microbiologíaRESUMEN
In forensic science, knowledge and understanding of material transfer and persistence is inherent to the interpretation of trace evidence and can provide vital information on the activity level surrounding a crime. Detecting metal ions in fingermark residue has long been of interest in the field of forensic science, due to the possibility of linking trace metal ion profiles to prior activity with specific metal objects (e.g. gun or explosive handling). Unfortunately, the imaging capability to visualise trace metal ions at sufficient spatial resolution to determine their distribution within a fingermark (micron level) was not previously available. Here, we demonstrate for the first time transfer and persistence of metals in fingermarks, at micron spatial resolution, using synchrotron sourced X-ray fluorescence microscopy. Such information may form a critical baseline for future metal-based detection strategies. Fingermarks were taken before and after brief handling of a gun barrel, ammunition cartridge case and party sparkler to demonstrate the transfer of metals. The results reveal increased metal content after contact with these objects, and critically, a differential pattern of metal ion increase was observed after handling different objects. Persistence studies indicate that these metals are removed as easily as they are transferred, with a brief period of hand washing appearing to successfully remove metallic residue from subsequent fingermarks. Preliminary work using X-ray absorption near edge structure spectroscopic mapping highlighted the potential use of this technique to differentiate between different chemical forms of metals and metal ions in latent fingermarks. It is anticipated that these findings can now be used to assist future work for the advancement of trace metal detection tests and fingermark development procedures.
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Dermatoglifia , Sustancias Explosivas , Ciencias Forenses , Metales , MicroscopíaRESUMEN
Degradation of fingermark residue has a major impact on the successful forensic detection of latent fingermarks. The time course of degradation has been previously explored with bulk chemical analyses, but little is known about chemical alterations within specific regions of the fingermark, which is difficult to study with bulk measurement. Here we report the use of synchrotron-sourced attenuated total reflection-Fourier transform infrared (ATR-FTIR) microspectroscopy to provide spatio-temporal resolution of chemical changes within fingermark droplets, as a function of time since deposition, under ambient temperature conditions. Eccrine and sebaceous material within natural fingermark droplets were imaged on the micron scales at hourly intervals from the time of deposition until the first 7-13 hours after deposition, revealing that substantial dehydration occurred within the first 8 hours. Changes to lipid material were more varied, with samples exhibiting an increase or decrease in lipid concentration due to the degradation and redistribution of this material. Across 12 donors, it was noticeable that the initial chemical composition and morphology of the droplet varied greatly, which appeared to influence the rate of change of the droplet over time. Further, this study attempted to quantify the total water content within fingermark samples. The wide-spread nature and strength of the absorption of Terahertz/Far-infrared (THz/Far-IR) radiation by water vapour molecules were exploited for this purpose, using THz/Far-IR gas-phase spectroscopy. Upon heating, water confined in natural fingermarks was evaporated and expanded in a vacuum chamber equipped with multipass optics. The amount of water vapour was then quantified by high-spectral resolution analysis, and fingermarks were observed to lose approximately 14-20 µg of water. The combination of both ATR-FTIR and Far-IR gas-phase techniques highlight important implications for experimental design in fingermark research, and operational practices used by law enforcement agencies.
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Dermatoglifia , Sincrotrones , Medicina Legal/métodos , Óptica y Fotónica , Espectrofotometría InfrarrojaRESUMEN
Magnetic resonance imaging (MRI) is the gold standard method to study brain anatomy in vivo. Using MRI, subtle alterations to white matter structures in the brain are observed prior to cognitive decline associated with the ageing process, and neurodegenerative diseases such as Alzheimer's disease. Detection of such alterations provides hope for early clinical diagnosis. While MRI is essential to detect subtle alterations to brain structure in vivo, the technique is less suited to study and image the distribution of biochemical markers within specific brain structures. Consequently, the chemical changes that drive, or are associated with MRI-detectable alterations to white matter are not well understood. Herein, we describe (to the best of our knowledge) the first application of a complementary imaging approach that incorporates in vivo MRI with ex vivo Fourier transform infrared (FTIR) spectroscopic imaging on the same brain tissue. The combined workflow is used to detect and associate markers of altered biochemistry (FTIR) with anatomical changes to brain white matter (MRI). We have applied this combination of techniques to the senescence accelerated murine prone strain 8 (SAMP8) mouse model (n = 6 animals in each group, analysed across two ageing time points, 6 and 12 months). The results have demonstrated alterations to lipid composition and markers of disturbed metabolism during ageing are associated with loss of white matter volume.
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Sustancia Blanca , Animales , Ratones , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Química Encefálica , Análisis de Fourier , Espectroscopía Infrarroja por Transformada de Fourier , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Imagen por Resonancia Magnética/métodos , Envejecimiento , NeuroimagenRESUMEN
Hepatocytes are essential for maintaining the homeostasis of iron and lipid metabolism in mammals. Dysregulation of either iron or lipids has been linked with serious health consequences, including non-alcoholic fatty liver disease (NAFLD). Considered the hepatic manifestation of metabolic syndrome, NAFLD is characterised by dysregulated lipid metabolism leading to a lipid storage phenotype. Mild to moderate increases in hepatic iron have been observed in â¼30% of individuals with NAFLD; however, direct observation of the mechanism behind this increase has remained elusive. To address this issue, we sought to determine the metabolic consequences of iron loading on cellular metabolism using live cell, time-lapse Fourier transform infrared (FTIR) microscopy utilising a synchrotron radiation source to track biochemical changes. The use of synchrotron FTIR is non-destructive and label-free, and allowed observation of spatially resolved, sub-cellular biochemical changes over a period of 8â h. Using this approach, we have demonstrated that iron loading in AML12 cells induced perturbation of lipid metabolism congruent with steatosis development. Iron-loaded cells had approximately three times higher relative ester carbonyl concentration compared with controls, indicating an accumulation of triglycerides. The methylene/methyl ratio qualitatively suggests the acyl chain length of fatty acids in iron-loaded cells increased over the 8â h period of monitoring compared with a reduction observed in the control cells. Our findings provide direct evidence that mild to moderate iron loading in hepatocytes drives de novo lipid synthesis, consistent with a role for iron in the initial hepatic lipid accumulation that leads to the development of hepatic steatosis.
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Rastreo Celular/métodos , Hígado Graso/patología , Hepatocitos/metabolismo , Sobrecarga de Hierro/fisiopatología , Hierro/metabolismo , Sincrotrones/instrumentación , Imagen de Lapso de Tiempo/métodos , Animales , Hígado Graso/metabolismo , Hepatocitos/citología , Metabolismo de los Lípidos , Ratones , MicroscopíaRESUMEN
Understanding the interaction of proteins at interfaces, which occurs at or within cell membranes and lipoprotein vesicles, is central to our understanding of protein function. Therefore, new experimental approaches to understand how protein structure is influenced by protein-interface interactions are important. Herein we build on our previous work exploring electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) to investigate changes in protein secondary structure that are modulated by protein-interface interactions. The ITIES provides an experimental framework to drive protein adsorption at an interface, allowing subsequent spectroscopic analysis (e.g., Fourier transform infrared spectroscopy) to monitor changes in protein structure. Here, we reveal that the interaction between insulin and the interface destabilizes native insulin secondary structure, promoting formation of α helix secondary structures. These structural alterations result from protein-interface rather than protein-protein interactions at the interface. Although this is an emerging approach, our results provide a foundation highlighting the value of the ITIES as a tool to study protein structure and interactions at interfaces. Such knowledge may be useful to elucidate protein function within biological systems or to aid sensor development.
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Insulina , Proteínas , Adsorción , Electroquímica , Estructura Secundaria de ProteínaRESUMEN
Metal homeostasis is integral to normal plant growth and development. During plant-pathogen interactions, the host and pathogen compete for the same nutrients, potentially impacting nutritional homeostasis. Our knowledge of outcome of the interaction in terms of metal homeostasis is still limited. Here, we employed the X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron to visualize and analyse the fate of nutrients in wheat leaves infected with Pyrenophora tritici-repentis, a necrotrophic fungal pathogen. We sought to (i) evaluate the utility of XFM for sub-micron mapping of essential mineral nutrients and (ii) examine the spatiotemporal impact of a pathogen on nutrient distribution in leaves. XFM maps of K, Ca, Fe, Cu, Mn, and Zn revealed substantial hyperaccumulation within, and depletion around, the infected region relative to uninfected control samples. Fungal mycelia were visualized as thread-like structures in the Cu and Zn maps. The hyperaccumulation of Mn in the lesion and localized depletion in asymptomatic tissue surrounding the lesion was unexpected. Similarly, Ca accumulated at the periphery of the symptomatic region and as microaccumulations aligning with fungal mycelia. Collectively, our results highlight that XFM imaging provides the capability for high-resolution mapping of elements to probe nutrient distribution in hydrated diseased leaves in situ.
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Nutrientes , Sincrotrones , Ascomicetos , Australia , Microscopía Fluorescente , Rayos XRESUMEN
Correction for 'Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells' by Jitraporn Vongsvivut et al., Analyst, 2019, 144, 3226-3238, DOI: 10.1039/C8AN01543K.
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Visualising direct biochemical markers of cell physiology and disease pathology at the sub-cellular level is an ongoing challenge in the biological sciences. A suite of microscopies exists to either visualise sub-cellular architecture or to indirectly view biochemical markers (e.g. histochemistry), but further technique developments and innovations are required to increase the range of biochemical parameters that can be imaged directly, in situ, within cells and tissue. Here, we report our continued advancements in the application of synchrotron radiation attenuated total reflectance Fourier transform infrared (SR-ATR-FTIR) microspectroscopy to study sub-cellular biochemistry. Our recent applications demonstrate the much needed capability to map or image directly sub-cellular protein aggregates within degenerating neurons as well as lipid inclusions within bacterial cells. We also characterise the effect of spectral acquisition parameters on speed of data collection and the associated trade-offs between a realistic experimental time frame and spectral/image quality. Specifically, the study highlights that the choice of 8 cm-1 spectral resolutions provide a suitable trade-off between spectral quality and collection time, enabling identification of important spectroscopic markers, while increasing image acquisition by â¼30% (relative to 4 cm-1 spectral resolution). Further, this study explores coupling a focal plane array detector with SR-ATR-FTIR, revealing a modest time improvement in image acquisition time (factor of 2.8). Such information continues to lay the foundation for these spectroscopic methods to be readily available for, and adopted by, the biological science community to facilitate new interdisciplinary endeavours to unravel complex biochemical questions and expand emerging areas of study.
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Agregado de Proteínas , Sincrotrones , Lípidos , Proteínas , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Background: A body of epidemiological, clinical and preclinical studies suggest increased risk for cerebro- and cardio-vascular disease associated with dietary ingestion of long-chain saturated fatty acids (LCSFA). In wild-type rodent models, chronic ingestion of LCSFA diets are associated with increased cerebral capillary permeability, heightened neurovascular inflammation and poorer cognitive performance. However, recent studies suggest that diets enriched in fat may paradoxically attenuate elements of the ageing phenotype via a caloric support axis.Objective: The purpose of this study was to explore the effects of dietary LCSFA on cerebral capillary integrity and neurovascular inflammation in an established model of accelerated ageing, Senescence-Accelerated-Murine-Prone Strain 8 (SAMP8) mice.Methods: From 6 weeks of age, SAMP8 mice and age-matched controls were randomised to either normal chow, or to an LCSFA-enriched diet, for either 12 or 34 weeks. An additional group of SAMP8 mice were provided the LCSFA-enriched diet for 12 weeks followed by the provision of ordinary low-fat chow for 22 weeks. Ex vivo measures of cerebrovascular integrity, neurovascular inflammation and astrocytic activation, were determined via 3-dimensional immunofluorescent confocal microscopy methodologies.Results: LCSFA-fed SAMP8 mice had markedly attenuated cerebral capillary dysfunction concomitant with reduced microglial activation. In SAMP8 mice transiently maintained on an LCSFA diet for 12 weeks, suppression of neurovascular inflammation persisted. Marked hippocampal astrogliosis was evident in LCSFA-fed mice when compared to SAMP8 mice maintained on ordinary chow.Conclusion: The findings from this study support the notion that high-fat, potentially ketogenic diets, may confer neuroprotection in SAMP8 mice through a vascular-support axis.
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Envejecimiento/fisiología , Corteza Cerebral/irrigación sanguínea , Dieta Alta en Grasa , Encefalitis/fisiopatología , Envejecimiento/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Ácidos Grasos/administración & dosificación , Masculino , RatonesRESUMEN
Studies show that vitamin D (vit-D) (25(OH)D), the bioactive metabolite (1,25(OH)2D3) and vit-D receptors (vit-D receptor; protein disulphide isomerase, family A member 3) are expressed throughout the brain, particularly in regions pivotal to learning and memory. This has led to the paradigm that avoiding vit-D deficiency is important to preserve cognitive function. However, presently, it is not clear if the common clinical measure of serum 25(OH)D serves as a robust surrogate marker for central nervous system (CNS) homeostasis or function. Indeed, recent studies report CNS biosynthesis of endogenous 25(OH)D, the CNS expression of the CYP group of enzymes which catalyse conversion to 1,25(OH)2D3 and thereafter, deactivation. Moreover, in the periphery, there is significant ethnic/genetic heterogeneity in vit-D conversion to 1,25(OH)2D3 and there is a paucity of studies which have actually investigated vit-D kinetics across the cerebrovasculature. Compared with peripheral organs, the CNS also has differential expression of receptors that trigger cellular response to 1,25(OH)2D3 metabolites. To holistically consider the putative association of peripheral (blood) abundance of 25(OH)D on cognitive function, herein, we have reviewed population and genetic studies, pre-clinical and clinical intervention studies and moreover have considered potential confounders of vit-D analysis.
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Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 µm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.
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Distrofia Muscular de Duchenne , Animales , Suplementos Dietéticos , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético , Sincrotrones , Taurina/farmacologíaRESUMEN
The effect of halogen substitution in fluorescent BODIPY species was evaluated in the context of staining lipids in situ within brain tissue sections. Herein we demonstrate that the halogenated species maintain their known in vitro affinity when applied to detect lipids in situ in brain tissue sections. Interestingly, the chlorine substituted compound revealed the highest specificify for white matter lipids. Furthermore, the halogen substituted compounds rapidly detected lipid enriched cells, in situ, associated with a case of brain pathology and neuroinflammation.
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Compuestos de Boro/química , Cerebelo/diagnóstico por imagen , Colorantes Fluorescentes/química , Animales , Halogenación , Accidente Cerebrovascular Isquémico/diagnóstico por imagen , Masculino , Ratones Endogámicos BALB C , Microscopía FluorescenteRESUMEN
Fingermarks are an important form of crime-scene trace evidence; however, their usefulness may be hampered by a variation in response or a lack of robustness in detection methods. Understanding the chemical composition and distribution within fingermarks may help explain variation in latent fingermark detection with existing methods and identify new strategies to increase detection capabilities. The majority of research in the literature describes investigation of organic components of fingermark residue, leaving the elemental distribution less well understood. The relative scarcity of information regarding the elemental distribution within fingermarks is in part due to previous unavailability of direct, micron resolution elemental mapping techniques. This capability is now provided at third generation synchrotron light sources, where X-ray fluorescence microscopy (XFM) provides micron or submicron spatial resolution and direct detection with sub-µM detection limits. XFM has been applied in this study to reveal the distribution of inorganic components within fingermark residue, including endogenous trace metals (Fe, Cu, Zn), diffusible ions (Cl-, K+, Ca2+), and exogeneous metals (Ni, Ti, Bi). This study incorporated a multimodal approach using XFM and infrared microspectroscopy analyses to demonstrate colocalization of endogenous metals within the hydrophilic organic components of fingermark residue. Additional experiments were then undertaken to investigate how sources of exogenous metals (e.g., coins and cosmetics) may be transferred to, and distributed within, latent fingermarks. Lastly, this study reports a preliminary assessment of how environmental factors such as exposure to aqueous environments may affect elemental distribution within fingermarks. Taken together, the results of this study advance our current understanding of fingermark composition and its spatial distribution of chemical components and may help explain detection variation observed during detection of fingermarks using standard forensic protocols.
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A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520â nm) to red (600â nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).
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Bacillus cereus/metabolismo , Complejos de Coordinación/química , Iridio/química , Lípidos/química , Microscopía Confocal/métodos , Complejos de Coordinación/metabolismo , Sustancias Luminiscentes/química , Espectrometría RamanRESUMEN
The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated antiparallel ß-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurements, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modeled the structure of lysozyme at an aqueous-organic interface. On the basis of these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.
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Citocromos c/química , Geles/química , Hemoglobinas/química , Muramidasa/química , Mioglobina/química , Adsorción , Animales , Boratos/química , Bovinos , Pollos , Técnicas Electroquímicas , Caballos , Simulación de Dinámica Molecular , Compuestos Organofosforados/química , Conformación Proteica en Lámina beta , Desplegamiento Proteico , Agua/químicaRESUMEN
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called "hybrid" macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 µm and 100 µm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science.