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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.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors ( tlr2 , tlr5 , tlr7, and tlr13 ), urokinase plasminogen activator surface receptor -like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1 . Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1 -like, macrophage mannose receptor-1 , crem , il-10 , and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.

Cyanobacterial blooms are often associated with the presence of harmful natural compounds which can cause adverse health effects in both humans and animals. One family of these compounds, known as anatoxins, have been linked to the rapid deaths of cattle and dogs through neurotoxicological action. Here, we report the findings resulting from the death of a dog at a freshwater reservoir in SW England. Poisoning was rapid following exposure to material at the side of the lake. Clinical signs included neurological distress, diaphragmatic paralysis and asphyxia prior to death after 45 min of exposure. Analysis by HILIC-MS/MS of urine and stomach content samples from the dog revealed the detection of anatoxin-a and dihydroanatoxin-a in both samples with higher concentrations of the latter quantified in both matrices. Detection and quantitative accuracy was further confirmed with use of accurate mass LC-HRMS. Additional anatoxin analogues were also detected by LC-HRMS, including 4-keto anatoxin-a, 4-keto-homo anatoxin-a, expoxy anatoxin-a and epoxy homo anatoxin-a. The conclusion of neurotoxicosis was confirmed with the use of two independent analytical methods showing positive detection and significantly high quantified concentrations of these neurotoxins in clinical samples. Together with the clinical signs observed, we have confirmed that anatoxins were responsible for the rapid death of the dog in this case.

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., (© 2022. The Author(s).)

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.

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.

There is increased recognition of the effects of diffuse traumatic brain injury (dTBI), which can initiate yet unknown biochemical cascades, resulting in delayed secondary brain degeneration and long-term neurological sequela. There is limited availability of therapies that minimize the effect of secondary brain damage on the quality of life of people who have suffered TBI, many of which were otherwise healthy adults. Understanding the cascade of biochemical events initiated in specific brain regions in the acute phase of dTBI and how this spreads into adjacent brain structures may provide the necessary insight into drive development of improved therapies. In this study, we have used direct biochemical imaging techniques (Fourier transform infrared spectroscopic imaging) and elemental mapping (X-ray fluorescence microscopy) to characterize biochemical and elemental alterations that occur in corpus callosum white matter in the acute phase of dTBI. The results provide direct visualization of differential biochemical and ionic changes that occur in the highly vulnerable medial corpus callosum white matter relative to the less vulnerable lateral regions of the corpus callosum. Specifically, the results suggest that altered ionic gradients manifest within mechanically damaged medial corpus callosum, potentially spreading to and inducing lipid alterations to white matter structures in lateral brain regions.

Diffusible ions (Na + , K + , Mg 2+ , Ca 2+ , Cl - ) are vital for healthy function of all cells, especially brain cells. Unfortunately, the diffusible nature of these ions renders them difficult to study with traditional microscopy in situ within ex vivo brain tissue sections. This mini-review examines the recent progress in the field, using direct elemental mapping techniques to study ion homeostasis during normal brain physiology and pathophysiology, through measurement of ion distribution and concentration in ex vivo brain tissue sections. The mini-review examines the advantages and limitations of specific techniques: proton induced X-ray emission (PIXE), X-ray fluorescence microscopy (XFM), secondary ion mass spectrometry (SIMS), laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and the sample preparation requirements to study diffusible ions with these methods., (Copyright © 2020 Hartnell, Andrews, Smith, Jiang, McAllum, Rajan, Colbourne, Fitzgerald, Lam, Takechi, Pushie, Kelly and Hackett.)

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.