A commentary on studies of brain iron accumulation during ageing.

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.

Elemental Mapping in a Preclinical Animal Model Reveals White Matter Copper Elevation in the Acute Phase of Central Nervous System Trauma.

Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.

Spatiotemporal patterns of wheat response to Pyrenophora tritici-repentis in asymptomatic regions revealed by transcriptomic and X-ray fluorescence microscopy analyses.

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.

Revisiting the Conformational Isomerism of Dihaloethanes: A Hybrid Computational and Experimental Laboratory for the Undergraduate Curriculum.

The conformational isomerism of disubstituted ethanes is a well-known concept that is part of every chemistry curriculum. Due to the species' simplicity, studying the (free) energy difference between the gauche and anti isomers has been the testing ground of experimental and computational techniques, such as Raman and IR spectroscopy, quantum chemistry, and atomistic simulations. While students normally receive formal training in spectroscopic techniques during their early undergraduate years, computational methods often receive less attention. In this work, we revisit the conformational isomerism of 1,2-dichloroethane and 1,2-dibromoethane and design a hybrid computational and experimental laboratory for our undergraduate chemistry curriculum with a focus on introducing computational techniques as a complementary research tool to experimentation. We show how commonly available Raman spectrometers and atomistic simulations performed on desktop computers can be combined to study the conformational isomerism of disubstituted ethanes while discussing the advantages and limitations of the different approaches.

Association between ageing, brain chemistry and white matter volume revealed with complementary MRI and FTIR brain imaging.

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.

Characterising murine hippocampal iron homeostasis, in relation to markers of brain inflammation and metabolism, during ageing.

Metal ions (Fe, Cu, and Zn) are essential to a healthy brain function, with the amount, localisation, and chemical form often tightly controlled. Evidence points towards loss of metal ion homeostasis within the ageing brain; in particular brain Fe accumulation appears to be a hallmark of ageing, which may place the brain at a greater risk of neurodegenerative disease. Unfortunately, the cause or consequence of altered brain metal ion homeostasis during ageing remains unknown, and there is a lack of data comparing brain metal ion homeostasis with other events of the ageing process (e.g. brain metabolism, brain inflammation). This study has utilised a multi-modal approach that incorporated: X-ray fluorescence microscopy for elemental mapping of metal ion homeostasis, Perl's Fe histochemistry, FTIR spectroscopic biochemical imaging of lactate and protein aggregates, and immuno-fluorescence analysis of markers of brain inflammation and Fe storage proteins (heavy-chain ferritin, light-chain ferritin, and mitochondrial ferritin). Interestingly, while age-related Fe accumulation was observed in corpus callosum white matter of murine (C56BL/6J) brain tissue (concomitant with elevated levels of markers of brain inflammation and altered metabolism), Fe content was not altered within the hippocampus (a decrease in total Zn within the mossy fibres was observed). Ultimately, the results of this study demonstrate an important association between elevated brain Fe and brain inflammation during natural ageing. This study also highlights that future research is required to image different chemical forms of Fe with respect to changes in brain metabolism and inflammation, as well as localising these changes to specific cell types.

Luminescent Metal Complexes as Emerging Tools for Lipid Imaging.

Fluorescence microscopy is a key tool in the biological sciences, which finds use as a routine laboratory technique (e.g., epifluorescence microscope) or more advanced confocal, two-photon, and super-resolution applications. Through continued developments in microscopy, and other analytical methods, the importance of lipids as constituents of subcellular organelles, signalling or regulating molecules continues to emerge. The increasing recognition of the importance of lipids to fundamental cell biology (in health and disease) has prompted the development of protocols and techniques to image the distribution of lipids in cells and tissues. A diverse suite of spectroscopic and microscopy tools are continuously being developed and explored to add to the "toolbox" to study lipid biology. A relatively recent breakthrough in this field has been the development and subsequent application of metal-based luminescent complexes for imaging lipids in biological systems. These metal-based compounds appear to offer advantages with respect to their tunability of the photophysical properties, in addition to capabilities centred around selectively targeting specific lipid structures or classes of lipids. The presence of the metal centre also opens the path to alternative imaging modalities that might not be applicable to traditional organic fluorophores. This review examines the current progress and developments in metal-based luminescent complexes to study lipids, in addition to exploring potential new avenues and challenges for the field to take.

X-ray fluorescence microscopy methods for biological tissues.

Synchrotron-based X-ray fluorescence microscopy is a flexible tool for identifying the distribution of trace elements in biological specimens across a broad range of sample sizes. The technique is not particularly limited by sample type and can be performed on ancient fossils, fixed or fresh tissue specimens, and in some cases even live tissue and live cells can be studied. The technique can also be expanded to provide chemical specificity to elemental maps, either at individual points of interest in a map or across a large field of view. While virtually any sample type can be characterized with X-ray fluorescence microscopy, common biological sample preparation methods (often borrowed from other fields, such as histology) can lead to unforeseen pitfalls, resulting in altered element distributions and concentrations. A general overview of sample preparation and data-acquisition methods for X-ray fluorescence microscopy is presented, along with outlining the general approach for applying this technique to a new field of investigation for prospective new users. Considerations for improving data acquisition and quality are reviewed as well as the effects of sample preparation, with a particular focus on soft tissues. The effects of common sample pretreatment steps as well as the underlying factors that govern which, and to what extent, specific elements are likely to be altered are reviewed along with common artifacts observed in X-ray fluorescence microscopy data.

Monitoring the chemical changes in fingermark residue over time using synchrotron infrared spectroscopy.

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.

The transfer and persistence of metals in latent fingermarks.

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.

A review of the "metallome" within neurons and glia, as revealed by elemental mapping of brain tissue.

It is now well established that transition metals, such as Iron (Fe), Copper (Cu), and Zinc (Zn) are necessary for healthy brain function. Although Fe, Cu, and Zn are essential to the brain, imbalances in the amount, distribution, or chemical form ("metallome") of these metals is linked to the pathology of numerous brain diseases or disorders. Despite the known importance of metal ions for both brain health and disease, the metallome that exists within specific types of brain cells is yet to be fully characterised. The aim of this mini-review is to present an overview of the current knowledge of the metallome found within specific brain cells (oligodendrocytes, astrocytes, microglia, and neurons), as revealed by direct elemental mapping techniques. It is hoped this review will foster continued research using direct elemental mapping techniques to fully characterise the brain cell metallome.

Synthesis of human amyloid restricted to liver results in an Alzheimer disease-like neurodegenerative phenotype.

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.

"Wax On, Wax Off": In Vivo Imaging of Plant Physiology and Disease with Fourier Transform Infrared Reflectance Microspectroscopy.

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well-established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non-destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change.

Structural Changes in Insulin at a Soft Electrochemical Interface.

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.

Correction: Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells.

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.

[Mapping the Metallo-maze to Memory Loss: Does Neuronal Metal Ion Deficiency Contribute to Dementia?]

Dementia has no cure and is an international health crisis. In addition to the immeasurable loss of QOL caused by dementia, the global economic cost is predicted to reach $2 trillion (USD) by 2030. Although much remains unknown about the biochemical pathways driving cognitive decline and memory loss during dementia, metals have been implicated in neurodegenerative disease. For example, total levels of Fe and Cu increase, which has been proposed to drive oxidative stress; and Fe, Cu, and Zn can bind amyloid-ß, catalysing aggregation and formation of amyloid plaques. Unfortunately, despite these known facets through which metal ions may induce pathology, studies in greater detail have been hampered by a lack of microscopy methods to directly visualise metal ions, and their chemical form, within brain cells. Herein we report the use of synchrotron X-ray fluorescence microscopy to simultaneously image Fe, Cu, and Zn within neurons in ex vivo brain tissue sections. Using animal models of dementia, we now demonstrate for the first time that despite global increases in brain metal content and metal ion accumulation within amyloid plaques, key brain regions may also become metal ion deficient. Such deficiency could contribute to cognitive decline because of the essential roles metal ions play in neurotransmitter synthesis and energy metabolism. These recent findings are discussed in the context of memory loss, and the impact that metal ion dis-homeostasis may have on diagnostic and therapeutic development.

Blood-brain barrier disruption and ventricular enlargement are the earliest neuropathological changes in rats with repeated sub-concussive impacts over 2 weeks.

Repeated sub-concussive impact (e.g. soccer ball heading), a significantly lighter form of mild traumatic brain injury, is increasingly suggested to cumulatively alter brain structure and compromise neurobehavioural function in the long-term. However, the underlying mechanisms whereby repeated long-term sub-concussion induces cerebral structural and neurobehavioural changes are currently unknown. Here, we utilised an established rat model to investigate the effects of repeated sub-concussion on size of lateral ventricles, cerebrovascular blood-brain barrier (BBB) integrity, neuroinflammation, oxidative stress, and biochemical distribution. Following repeated sub-concussion 3 days per week for 2 weeks, the rats showed significantly enlarged lateral ventricles compared with the rats receiving sham-only procedure. The sub-concussive rats also presented significant BBB dysfunction in the cerebral cortex and hippocampal formation, whilst neuromotor function assessed by beamwalk and rotarod tests were comparable to the sham rats. Immunofluorescent and spectroscopic microscopy analyses revealed no significant changes in neuroinflammation, oxidative stress, lipid distribution or protein aggregation, within the hippocampus and cortex. These data collectively indicate that repeated sub-concussion for 2 weeks induce significant ventriculomegaly and BBB disruption, preceding neuromotor deficits.

Mapping sub-cellular protein aggregates and lipid inclusions using synchrotron ATR-FTIR microspectroscopy.

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 ∼30% (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.

Tracking biochemical changes induced by iron loading in AML12 cells with synchrotron live cell, time-lapse infrared microscopy.

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 ∼30% 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 8 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 8 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.