Multielement Z-tag imaging by X-ray fluorescence microscopy for next-generation multiplex imaging.

Rapid, highly multiplexed, nondestructive imaging that spans the molecular to the supra-cellular scale would be a powerful tool for tissue analysis. However, the physical constraints of established imaging methods limit the simultaneous improvement of these parameters. Whole-organism to atomic-level imaging is possible with tissue-penetrant, picometer-wavelength X-rays. To enable highly multiplexed X-ray imaging, we developed multielement Z-tag X-ray fluorescence (MEZ-XRF) that can operate at kHz speeds when combined with signal amplification by exchange reaction (SABER)-amplified Z-tag reagents. We demonstrated parallel imaging of 20 Z-tag or SABER Z-tag reagents at subcellular resolution in cell lines and multiple human tissues. We benchmarked MEZ-XRF against imaging mass cytometry and demonstrated the nondestructive multiscale repeat imaging capabilities of MEZ-XRF with rapid tissue overview scans, followed by slower, more sensitive imaging of low-abundance markers such as immune checkpoint proteins. The unique multiscale, nondestructive nature of MEZ-XRF, combined with SABER Z-tags for high sensitivity or enhanced speed, enables highly multiplexed bioimaging across biological scales.

The spatiotemporal spread of cervical spinal cord contusion injury pathology revealed by 3D in-line phase contrast synchrotron X-ray microtomography.

Extensive structural changes occur within the spinal cord following traumatic injury. Acute tissue debris and necrotic tissue are broken down, proliferating local glia and infiltrating leukocytes remodel tissue biochemical and biophysical properties, and a chronic cavity surrounded by a scar forms at the injury epicentre. Serial-section 2D histology has traditionally assessed these features in experimental models of spinal cord injury (SCI) to measure the extent of tissue pathology and evaluate efficacy of novel therapies. However, this 2D snapshot approach overlooks slice intervening features, with accurate representation of tissue compromised by mechanical processing artefacts. 3D imaging avoids these caveats and allows full exploration of the injured tissue volume to characterise whole tissue pathology. Amongst 3D imaging modalities, Synchrotron Radiation X-ray microtomography (SRµCT) is advantageous for its speed, ability to cover large tissue volumes at high resolution, and need for minimal sample processing. Here we demonstrate how extended lengths of formalin-fixed, paraffin-embedded (FFPE) rat spinal cord can be completely imaged by SRµCT with micron resolution. Label-free contrast derived from X-ray phase interactions with low-density soft tissues, reveals spinal cord white matter, gray matter, tissue damage and vasculature, with tissue still viable for targeted 2D-histology after 3D imaging. We used SRµCT to quantify tissue pathology after a midline, cervical level (C6), 225 kDyne contusion injury over acute-to-chronic (24 h to 5 weeks) post injury time points. Quantification revealed acute tissue swelling prior to chronic atrophy across the whole imaged region (spanning 2 spinal segments above and below injury), along with rostro-caudal asymmetries in white and gray matter volume loss. 3D volumes revealed satellite damage in tissue far removed from the epicentre, and extensive rostro-caudal spread of damage through the base of the dorsal columns at 24 h post injury. This damage overlapped regions of vasogenic oedema, confirmed with subsequent histology. Tissue damage at later time points in border regions was most prominent in the dorsal columns, where it overlapped sites of damaged venous vasculature. Elaborating rostro-caudal and spatiotemporal asymmetries in reduced traumatic injury models centred on these regions may inform future treatments that seek to limit the spread of tissue pathology to these 'at-risk' regions.

Optimising complementary soft tissue synchrotron X-ray microtomography for reversibly-stained central nervous system samples.

Synchrotron radiation microtomography (SRµCT) is a nominally non-destructive 3D imaging technique which can visualise the internal structures of whole soft tissues. As a multi-stage technique, the cumulative benefits of optimising sample preparation, scanning parameters and signal processing can improve SRµCT imaging efficiency, image quality, accuracy and ultimately, data utility. By evaluating different sample preparations (embedding media, tissue stains), imaging (projection number, propagation distance) and reconstruction (artefact correction, phase retrieval) parameters, a novel methodology (combining reversible iodine stain, wax embedding and inline phase contrast) was optimised for fast (~12 minutes), high-resolution (3.2-4.8 µm diameter capillaries resolved) imaging of the full diameter of a 3.5 mm length of rat spinal cord. White-grey matter macro-features and micro-features such as motoneurons and capillary-level vasculature could then be completely segmented from the imaged volume for analysis through the shallow machine learning SuRVoS Workbench. Imaged spinal cord tissue was preserved for subsequent histology, establishing a complementary SRµCT methodology that can be applied to study spinal cord pathologies or other nervous system tissues such as ganglia, nerves and brain. Further, our 'single-scan iterative downsampling' approach and side-by-side comparisons of mounting options, sample stains and phase contrast parameters should inform efficient, effective future soft tissue SRµCT experiment design.

Unique pharmacology of heteromeric α7ß2 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

α7ß2 is a novel type of nicotinic acetylcholine receptor shown to be uniquely expressed in cholinergic neurons of the basal forebrain and in hippocampal interneurons. We have compared the pharmacological properties of recombinant homomeric α7 and heteromeric α7ß2 nicotinic acetylcholine receptors in order to reveal the pharmacological consequences of ß2 subunit incorporation into the pentamer. The non-selective agonist epibatidine did not distinguish α7ß2 from α7 nicotinic acetylcholine receptors, but three other non-selective agonists (nicotine, cytisine and varenicline) were less efficacious on α7ß2 than on α7. A more dramatic change in efficacy was seen with eight different selective α7 agonists. Because of their very low intrinsic efficacy, some compounds became very efficacious functional antagonists at α7ß2 receptors. Three α4ß2 nicotinic receptor selective agonists that were not active on α7, were also inactive on α7ß2, and dihydro-ß-erythroidine, an α4ß2 receptor-preferring antagonist, inhibited α7 and α7ß2 in a similar manner. These results reveal significant effects of ß2 incorporation in determining the relative efficacy of several non-selective and α7 selective agonists, and also show that incorporation of ß2 subunits does not cause a shift to a more "ß2-like" pharmacology of α7 nicotinic acetylcholine receptors.