Browsing by Author "Hackett, MJ"
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- ItemChemical alterations to murine brain tissue induced by formation fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis(Royal Society of Chemistry, 2011-07-21) Hackett, MJ; McQuillan, JA; El-Assaad, F; Aitken, JB; Levina, A; Cohen, DD; Siegele, R; Carter, EA; Grau, GE; Hunt, NH; Lay, PAUnderstanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture'' of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.© 2011, Royal Society of Chemistry
- ItemInvestigating the role of Zn in glucose regulation using x-ray fluorescence microscopy and x-ray absorption near-edge structure spectroscopy(Australian Nuclear Science and Technology Organisation, 2021-11-24) Ellison, G; Bambery, KR; Hackett, MJ; Hollings, A; Howard, DJ; Sharif, A; Takechi, RNZinc plays an important function in glucose regulation, particularly within pancreatic islets, the anatomical home of the glucose regulating hormones insulin and glucagon. Glucose dysregulation is a significant contributor to the epidemic of metabolic diseases, including diabetes, that affect an increasing number of people. Zn is found in very high (mM) concentrations in insulin-secreting β-cells, where it facilitates insulin synthesis and storage, and is co-secreted with insulin, subsequently acting as a signalling molecule. Zn dysregulation is often coincident with impairment of insulin secretion, but little is known about the nature of the changes. Since a subset of the pool of Zn in islets is labile, it is difficult to image in its in vivo situation using conventional techniques such as histochemistry. Not only do preparation steps such as washing displace Zn, but some forms in which it exists are not readily discernible using conventional microscopy techniques. X-ray fluorescence microscopy (XFM) and X-ray absorption near-edge structure spectroscopy (XANES) offer several advantages in that tissue preparation is minimal, facilitating the conservation of native states, and all forms of Zn are not only detectable, but are able to be discriminated by matching spectra against an existing library of Zn forms. Here we report the preliminary results from our study of Zn speciation and elemental mapping in murine islets from healthy or diabetes-prone animals in two age groups, 14 (denoted young) or 28 (old) weeks. This work uses a library of biologically relevant Zn forms created in our laboratory, and contributes to our understanding of the role of Zn in glucose regulation in health and disease, including aging. © The Authors
- ItemInvestigation of the mouse cerebellum using STIM and mu-PIXE spectrometric and FTIR spectroscopic mapping and imaging(Elsevier, 2011-10-15) Hackett, MJ; Siegele, R; El-Assaad, F; McQuillan, JA; Aitken, JB; Carter, EA; Grau, GE; Hunt, NH; Cohen, DD; Lay, PAThe cerebral biochemistry associated with the development of many neurological diseases remains poorly understood. In particular, incomplete understanding of the mechanisms through which vascular inflammation manifests in tissue damage and altered brain function is a significant hindrance to the development of improved patient therapies. To this extent, a combination of spectrometric/spectroscopic mapping/imaging methods with an inherent ability to provide a wealth of biochemical and physical information have been investigated to understand further the pathogenesis of brain disease. In this study, proton-induced X-ray emission (PIXE) mapping was combined with scanning transmission ion microscopy (STIM) mapping and Fourier-transform infrared (FTIR) imaging of the same tissue sample to study directly the composition of the murine (mouse) cerebellum. The combination of the elemental, density and molecular information provided by these techniques enabled differentiation between four specific tissue types within the murine cerebellum (grey matter, white matter, molecular layer and micro blood vessels). The results presented are complementary, multi-technique measurements of the same tissue sample. They show elemental, density and molecular differences among the different tissue types. (C) 2011 Elsevier B.V.
- ItemLight and heavy ion beam analysis of thin biological sections(Elsevier, 2013-07-01) Lee, J; Siegele, R; Pastuovic, Z; Hackett, MJ; Hunt, NH; Grau, GE; Cohen, DD; Lay, PAThe application of ion beam analysis (IBA) techniques to thin biological sections (ThBS) presents unique challenges in sample preparation, data acquisition and analysis. These samples are often the end product of expensive, time-consuming experiments, which involve many steps that require careful attention. Analysis via several techniques can maximise the information that is collected from these samples. Particle-induced X-ray emission (PIXE) and Rutherford backscattering (RBS) spectroscopy are two generally non-destructive IBA techniques that use the same MeV ions and can be performed simultaneously. The use of heavy ion PIXE applied to thick samples has, in the past, resulted in X-ray spectra of a poorer quality when compared to those obtained with proton beams. One of the reasons for this is the shorter probing depth of the heavy ions, which does not affect thin sample analysis. Therefore, we have investigated and compared 3-MeV proton and 36-MeV carbon ion beams on 7-μm thick mouse brain sections at the ANSTO Heavy ion microprobe (HIMP). The application of a 36-MeV C4+ ion beam for PIXE mapping of ThBS on thin Si3N4 substrate windows produced spectra of high quality that displayed close to a nine-times gain in signal yield (Z2/q) when compared to those obtained for 3-MeV protons for P, S, Cl and K but not for Fe, Cu and Zn. Image quality was overall similar; however, some elements showed better contrast and features with protons whilst others showed improved contrast with a carbon ion beam. RBS spectra with high enough counting statistics were easily obtained with 3-MeV proton beams resulting in high resolution carbon maps, however, the count rate for nitrogen and oxygen was too low. The results demonstrate that on thin samples, 36-MeV C4+ will produce good quality PIXE spectra in less time; therefore, carbon ions may be advantageous depending on which element is being studied. However, these advantages may be outweighed by the inherent disadvantages including increased ion beam damage, the necessity of very high ion energies resulting in higher neutron fields. © 2013, Elsevier B.V.
- ItemMechanisms of murine cerebral malaria: multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites(American Association for the Advancement of Science, 2015-12-18) Hackett, MJ; Aitken, JB; El-Assaad, F; McQuillan, JA; Carter, EA; Ball, HJ; Tobin, MJ; Paterson, DJ; de Jonge, MD; Siegele, R; Cohen, DD; Vogt, S; Grau, GE; Hunt, NH; Lay, PAUsing a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe–free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies. 2015 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution Non Commercial Licence 4.0 (CC BY-NC).
- ItemSpatiotemporal patterns of wheat response to Pyrenophora tritici-repentis in asymptomatic regions revealed by transcriptomic and x-ray fluorescence microscopy analyses(Oxford University Press (OUP), 2023-08-17) Moolhuijzen, P; Sanglard, LMVP; Paterson, DJ; Gray, SA; Khambatta, K; Hackett, MJ; Zerihun, A; Gibberd, MR; Naim, FPathogen 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. © The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
- ItemSpectroscopic analysis of age-related changes in the brain lateral ventricles during ageing(Australian Nuclear Science and Technology Organisation, 2021-11-24) Hollings, A; Hackett, MJ; Tobin, MJ; Klein, AR; Vongsvivut, JP; de Jonge, MD; Bone, S; Webb, S; Lam, V; Takechi, R; Mamo, JAlzheimer’s disease is the most common form of dementia and poses significant health and economic concerns. Currently, the disease has no cure, and it is expected that over 1 million people could be affected by 2058 in Australia alone. The content and distribution of metals such as Fe, Cu, Zn is known to change in the ageing brain and thus, increased understanding of the mechanistic role of metal dis-homeostasis may illuminate new therapeutic strategies. The brain lateral ventricles, which play a role in controlling metal and ion transport, have shown increasing levels of copper surrounding their walls with ageing. As a redox active metal, copper can induce oxidative stress which is a process that occurs during Alzheimer’s disease onset and progression. Our research group has been interested in determining whether the age-related elevation of copper surrounding the lateral ventricles is inducing oxidative stress in that region. In this study, we have utilised X-Ray Absorption Spectroscopy (XAS) at the Stanford Synchrotron Radiation Lightsource to analyse different chemical forms of sulfur and measure oxidative stress by analysis of disulfides. Additionally, we used the infrared microscopy beamline at the Australian Synchrotron to identify whether any other markers of oxidative stress were present around the ventricles. Further insights into metal dis-homeostasis and its influence on other biochemical pathways, may help to reveal some of the neurochemical mechanisms involved in progression of Alzheimer’s disease. In turn, this may help pave the way for potential preventative or therapeutic models.
- ItemSynchrotron x-ray fluorescence microscopy-enabled elemental mapping illuminates the ‘battle for nutrients’ between plant and pathogen(Oxford University Press, 2021-03-29) Naim, F; Khambatta, K; Sanglard, LMVP; Sauzier, G; Reinhardt, J; Paterson, DJ; Zerihun, A; Hackett, MJ; Gibberd, MRMetal 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. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.