Measuring Concentration of Nanoparticles in Polydisperse Mixtures Using Interferometric Nanoparticle Tracking Analysis.

Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion.

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.

Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells.

Bright-field light microscopy and related phase-sensitive techniques play an important role in life sciences because they provide facile and label-free insights into biological specimens. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in many high-end quantitative studies. Here, we demonstrate that interferometric scattering (iSCAT) microscopy operated in the confocal mode provides unique label-free solutions for live-cell studies. We reveal the nanometric topography of the nuclear envelope, quantify the dynamics of the endoplasmic reticulum, detect single microtubules, and map nanoscopic diffusion of clathrin-coated pits undergoing endocytosis. Furthermore, we introduce the combination of confocal and wide-field iSCAT modalities for simultaneous imaging of cellular structures and high-speed tracking of nanoscopic entities such as single SARS-CoV-2 virions. We benchmark our findings against simultaneously acquired fluorescence images. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes. The method is ideally suited for live studies on primary cells that face labeling challenges and for very long measurements beyond photobleaching times.

Pollen DNA metabarcoding reveals cryptic diversity and high spatial turnover in alpine plant-pollinator networks.

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks.

Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response.

All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.

Genotypic identification of Panicum spp. in New South Wales, Australia using DNA barcoding.

Australia has over 30 Panicum spp. (panic grass) including several non-native species that cause crop and pasture loss and hepatogenous photosensitisation in livestock. It is critical to correctly identify them at the species level to facilitate the development of appropriate management strategies for efficacious control of Panicum grasses in crops, fallows and pastures. Currently, identification of Panicum spp. relies on morphological examination of the reproductive structures, but this approach is only useful for flowering specimens and requires significant taxonomic expertise. To overcome this limitation, we used multi-locus DNA barcoding for the identification of ten selected Panicum spp. found in Australia. With the exception of P. buncei, other native Australian Panicum were genetically separated at the species level and distinguished from non-native species. One nuclear (ITS) and two chloroplast regions (matK and trnL intron-trnF) were identified with varying facility for DNA barcode separation of the Panicum species. Concatenation of sequences from ITS, matK and trnL intron-trnF regions provided clear separation of eight regionally collected species, with a maximum intraspecific distance of 0.22% and minimum interspecific distance of 0.33%. Two of three non-native Panicum species exhibited a smaller genome size compared to native species evaluated, and we speculate that this may be associated with biological advantages impacting invasion of non-native Panicum species in novel locations. We conclude that multi-locus DNA barcoding, in combination with traditional taxonomic identification, provides an accurate and cost-effective adjunctive tool for further distinguishing Panicum spp. at the species level.

Implications of the 2019-2020 megafires for the biogeography and conservation of Australian vegetation.

Australia's 2019-2020 'Black Summer' bushfires burnt more than 8 million hectares of vegetation across the south-east of the continent, an event unprecedented in the last 200 years. Here we report the impacts of these fires on vascular plant species and communities. Using a map of the fires generated from remotely sensed hotspot data we show that, across 11 Australian bioregions, 17 major native vegetation groups were severely burnt, and up to 67-83% of globally significant rainforests and eucalypt forests and woodlands. Based on geocoded species occurrence data we estimate that >50% of known populations or ranges of 816 native vascular plant species were burnt during the fires, including more than 100 species with geographic ranges more than 500 km across. Habitat and fire response data show that most affected species are resilient to fire. However, the massive biogeographic, demographic and taxonomic breadth of impacts of the 2019-2020 fires may leave some ecosystems, particularly relictual Gondwanan rainforests, susceptible to regeneration failure and landscape-scale decline.

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus' Lateral Body Protein.

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-ß promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes.

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

Author Correction: Quantitative mapping and minimization of super-resolution optical imaging artifacts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Findings from machine learning in clinical medical imaging applications - Lessons for translation to the forensic setting.

Machine learning (ML) techniques are increasingly being used in clinical medical imaging to automate distinct processing tasks. In post-mortem forensic radiology, the use of these algorithms presents significant challenges due to variability in organ position, structural changes from decomposition, inconsistent body placement in the scanner, and the presence of foreign bodies. Existing ML approaches in clinical imaging can likely be transferred to the forensic setting with careful consideration to account for the increased variability and temporal factors that affect the data used to train these algorithms. Additional steps are required to deal with these issues, by incorporating the possible variability into the training data through data augmentation, or by using atlases as a pre-processing step to account for death-related factors. A key application of ML would be then to highlight anatomical and gross pathological features of interest, or present information to help optimally determine the cause of death. In this review, we highlight results and limitations of applications in clinical medical imaging that use ML to determine key implications for their application in the forensic setting.

Mechanisms behind persistence of a fire-sensitive alternative stable state system in the Gibson Desert, Western Australia.

Alternative vegetation types that switch from one to another under contrasting fire regimes are termed fire-mediated alternative stable states (FMASS). Typically, pyrophylic communities (i.e., vegetation assemblages favored by burning) dominate under high frequencies or intensities of fire. Conversely, fire-sensitive (pyrophobic) vegetation types persist under long fire-free conditions. As the persistence traits of plants of FMASS systems are generally poorly researched, threshold levels of pyric disturbance that trigger 'state-switching' are often unknown. Dense thickets of the obligate-seeder shrub waputi (Aluta maisonneuvei ssp. maisonneuvei [Myrtaceae]) form fire-retarding woody islands within highly flammable spinifex (Triodia spp.) grasslands in arid Australia. To examine the tolerance of Aluta thickets to burning, we investigated: (1) the influence of post-fire rainfall and fire severity on recruitment (a field study); (2) soil seedbank densities (a field study); and (3) fire-related dormancy cues in seeds (a germination trial). We found a positive relationship between recruitment and post-fire rainfall volume, and much higher mean recruitment at sites with high- (5.9 seedlings/m2) than low-severity-burnt (2.2 seedlings/m2) and unburnt shrubs (0.03 seedlings/m2). Post-fire regeneration was mediated by dense soil-borne seedbanks, and the germination trial indicated that smoke promoted germination. Although Aluta shrubs are invariably fire-killed, high-severity fires are unlikely to lead to state shifts from shrubland to grassland because of the ability of mature stands to regenerate from dense, fire-cued seedbanks. Nevertheless, given that Aluta seedlings are exceptionally slow-growing, post-fire droughts combined with fire-return intervals less than the Aluta primary juvenile period of c. 5 years could drive conversion from Aluta- to Triodia-dominated vegetation.

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Super-resolution Microscopy of Vaccinia Virus Particles.

Super-resolution microscopy enables the study of vaccinia architecture at subviral resolution with molecular specificity. Here, we outline how to use structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM) to detect fluorescently tagged or immunolabeled viral proteins on purified virions. Tens to hundreds of individual virions can be imaged in a single field of view providing data for single-particle averaging or quantitative analysis of viral protein spatial organization.

Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation.

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

Development of a simple numerical model for trabecular bone structures.

PURPOSE: Advances in additive manufacturing processes are enabling the fabrication of surrogate bone structures for applications including use in high-resolution anthropomorphic phantoms. In this research, a simple numerical model is proposed that enables the generation of microarchitecture with similar statistical distribution to trabecular bone. METHODS: A human humerus, radius, ulna, and several vertebrae were scanned on the Imaging and Medical beamline at the Australian Synchrotron and the proposed numerical model was developed through the definition of two complex functions that encode the trabecular thickness and position-dependant spacing to generate volumetric surrogate trabecular structures. The structures reproduced those observed at 19 separate axial locations through the experimental bone volumes. The applicability of the model when incorporating a two-material approximation to absorption- and phase-contrast CT was also investigated through simulation. RESULTS: The synthetic structures, when compared with the real trabecular microarchitecture, yielded an average mean thickness error of 2 µm, and a mean difference in standard deviation of 33 µm for the humerus, 24 µm for the ulna and radius, and 15 µm for the vertebrae. Simulated absorption- and propagation-based phase contrast CT projection data were generated and reconstructed using the derived mathematical simplifications from the two-material approximation, and the phase-contrast effects were successfully demonstrated. CONCLUSIONS: The presented model reproduced trabecular distributions that could be used to generate phantoms for quality assurance and validation processes. The implication of utilizing a two-material approximation results in simplification of the additive manufacturing process and the generation of synthetic data that could be used for training of machine learning applications.

NanoJ: a high-performance open-source super-resolution microscopy toolbox.

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ-a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

Effect of external fixation rod coupling in computed tomography.

External fixation is a common tool in the treatment of complex fractures, correction of limb deformity, and salvage arthrodesis. These devices typically incorporate radio-opaque metal rods/struts connected at varying distances and orientations between rings. Whilst the predominant imaging modality is plain film radiology, computed tomography (CT) may be performed in order for the surgeon to make a more confident clinical decision (e.g. timing of frame removal, assessment of degree of arthrodesis). We used a fractured sheep leg to systematically assess CT imaging performance with a Discovery CT750 HD CT scanner (GE Healthcare) to show how rod coupling in both traditional Ilizarov and hexapod frames distorts images. We also investigated the role of dual-energy CT (DECT) and metal artefact reduction software (MARS) on the visualisation of the fractured leg. Whilst mechanical reasons predominantly dictate the rod/strut configurations when building a circular frame, rod coupling in CT can be minimised. Firstly, ideally, all or all but one rod can be removed during imaging resulting in no rod coupling. If this is not possible, strategies for configuring the rods to minimise the effect of the rod coupling on the region of interest are demonstrated, e.g., in the case of a four-rod construct, switching the two anterior rods to a more central single one will achieve this goal without particularly jeopardising mechanical strength for a short period. It is also shown that the addition of DECT and MARS results in a reduction of artefacts, but also affects tissue and bone differentiation.

Revealing Compartmentalized Diffusion in Living Cells with Interferometric Scattering Microscopy.

The spatiotemporal organization and dynamics of the plasma membrane and its constituents are central to cellular function. Fluorescence-based single-particle tracking has emerged as a powerful approach for studying the single molecule behavior of plasma-membrane-associated events because of its excellent background suppression, at the expense of imaging speed and observation time. Here, we show that interferometric scattering microscopy combined with 40 nm gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons with up to 3 nm precision and 25 µs temporal resolution. The achievable spatiotemporal precision enabled us to reveal signatures of compartmentalization in neurons likely caused by the actin cytoskeleton.

Quantitative mapping and minimization of super-resolution optical imaging artifacts.

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.