Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy.

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 µm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.

A proxy for oxygen storage capacity from high-throughput screening and automated data analysis.

Oxygen storage and release is a foundational part of many key pathways in heterogeneous catalysis, such as the Mars-van Krevelen mechanism. However, direct measurement of oxygen storage capacity (OSC) is time-consuming and difficult to parallelise. To accelerate the discovery of stable high OSC rare-earth doped ceria-zirconia oxygen storage catalysts, a high-throughput robotic-based co-precipitation synthesis route was coupled with sequentially automated powder X-ray diffraction (PXRD), Raman and thermogravimetric analysis (TGA) characterisation of the resulting materials libraries. Automated extraction of data enabled rapid trend identification and provided a data set for the development of an OSC prediction model, investigating the significance of each extracted quantity towards OSC. The optimal OSC prediction model produced incorporated variables from only fast-to-measure analytical techniques and gave predicted values of OSC that agreed with experimental observations across an independent validation set. Those measured quantities that feature in the model emerge as proxies for OSC performance. The ability to predict the OSC of the materials accelerates the discovery of high-capacity oxygen storage materials and motivates the development of similar high-throughput workflows to identify candidate catalysts for other heterogeneous transformations.

In situ real-time neutron imaging of gaseous H2 adsorption and D2 exchange on carbon-supported Pd catalysts.

We use in situ neutron imaging to observe the adsorption/absorption of hydrogen within a packed catalyst bed of a Pd/C catalyst at a spatial and temporal resolution of ∼430 µm and a ∼9 s respectively. Additionally, the H2/D2 exchange process across the catalyst bed is followed in real time.

Extending fluorescence anisotropy to large complexes using reversibly switchable proteins.

The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1-30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.

Peroxisome proliferator-activated receptor γ coactivator 1-α overexpression improves angiogenic signalling potential of skeletal muscle-derived extracellular vesicles.

NEW FINDINGS: What is the central question of this study? Skeletal muscle extracellular vesicles likely act as pro-angiogenic signalling factors: does overexpression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) alter skeletal muscle myotube extracellular vesicle release, contents and angiogenic potential? What is the main finding and its importance? Overexpression of PGC-1α results in secretion of extracellular vesicles that elevate measures of angiogenesis and protect against acute oxidative stress in vitro. Skeletal muscle with high levels of PGC-1α expression, commonly associated with exercise induced angiogenesis and high basal capillarization, may secrete extracellular vesicles that support capillary growth and maintenance. ABSTRACT: Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM-EVs). Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC-1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC-1α regulates SkM-EV release, contents and angiogenic signalling potential. PGC-1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC-1α-overexpressing myotubes (PGC-EVs) as well as from green fluorescent protein-overexpressing myotubes (GFP-EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC-1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2-related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC-EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (-41%) compared to GFP-EVs. Additionally, PGC-EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC-1α in human myotubes increases the angiogenic potential of SkM-EVs. These angiogenic benefits coincided with increased anti-oxidative capacity of recipient HUVECs. High PGC-1α expression in skeletal muscle may prompt the release of SkM-EVs that support vascular redox homeostasis and angiogenesis.

DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 µm axially over an imaging volume of 3,000 × 800 × 300 µm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.

Increasing the field-of-view in oblique plane microscopy via optical tiling.

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe a large field of view in a dimensionality reduced format, identify features of interest, and volumetrically image these regions with high spatiotemporal resolution.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Nuclear spin relaxation as a probe of zeolite acidity: a combined NMR and TPD investigation of pyridine in HZSM-5.

The relative surface affinities of pyridine within microporous HZSM-5 zeolites are explored using two-dimensional 1H nuclear magnetic resonance (NMR) relaxation time measurements. The dimensionless ratio of longitudinal-to-transverse nuclear spin relaxation times T1/T2 is shown to exhibit strong sensitivity to the silica/alumina ratio (SAR) of these zeolites, which is indicative of material acidity. This trend is interpreted in terms of increased pyridine surface affinity with decreasing SAR. Temperature programmed desorption (TPD) analysis corroborates this observation, revealing a distinct increase in the heat of desorption associated with adsorbed pyridine as a function of decreasing SAR. A direct correlation between NMR and TPD data suggests NMR relaxation time analysis can be a valuable tool for the non-invasive characterisation of adsorption phenomena in microporous solids.

Operando XAFS investigation on the effect of ash deposition on three-way catalyst used in gasoline particulate filters and the effect of the manufacturing process on the catalytic activity.

Platinum group metals such as palladium and rhodium based catalysts are currently being implemented in gasoline particulate filter (GPF) autoexhaust after treatment systems. However, little is known about how the trapped particulate matter, such as the incombustible ash, interacts with the catalyst and so may affect its performance. Thisoperandostudy follows the evolution of the Pd found in two different model GPF systems: one containing ash components extracted from a GPF and another from a catalyst washcoat prior to adhesion onto the GPF. We show that the catalytic activity of the two systems vary when compared with a 0 g ash containing GPF. Compared to the 0 g ash sample the 20 g ash containing sample had a higher CO light off temperature, in addition, an oscillation profile for CO, CO2and O2was observed, which is speculated to be a combination of CO oxidation, C deposition via a Boudouard reaction and further partial oxidation of the deposited species to CO. During the ageing procedure the washcoat sample reduces NO at a lower temperature than the 0 g ash sample. However, post ageing the 0 g ash sample recovers and both samples reduce NO at 310 °C. In comparison, the 20 g ash GPF sample maintains a higher NO reduction temperature of 410 °C post ageing, implying that the combination of high temperature ageing and presence of ash has an irreversible negative effect on catalyst performance.

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.

4D In-Situ Microscopy of Aerosol Filtration in a Wall Flow Filter.

The transient nature of the internal pore structure of particulate wall flow filters, caused by the continuous deposition of particulate matter, makes studying their flow and filtration characteristics challenging. In this article we present a new methodology and first experimental demonstration of time resolved in-situ synchrotron micro X-ray computed tomography (micro-CT) to study aerosol filtration. We directly imaged in 4D (3D plus time) pore scale deposits of TiO2 nanoparticles (nominal mean primary diameter of 25 nm) with a pixel resolution of 1.6 µm. We obtained 3D tomograms at a rate of ∼1 per minute. The combined spatial and temporal resolution allows us to observe pore blocking and filling phenomena as they occur in the filter's pore space. We quantified the reduction in filter porosity over time, from an initial porosity of 0.60 to a final porosity of 0.56 after 20 min. Furthermore, the penetration depth of particulate deposits and filtration rate was quantified. This novel image-based method offers valuable and statistically relevant insights into how the pore structure and function evolves during particulate filtration. Our data set will allow validation of simulations of automotive wall flow filters. Evolutions of this experimental design have potential for the study of a wide range of dry aerosol filters and could be directly applied to catalysed automotive wall flow filters.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Predicting resolution and image quality in RESOLFT and other point scanning microscopes [Invited].

The performance of fluorescence microscopy and nanoscopy is often discussed by the effective point spread function and the optical transfer function. However, due to the complexity of the fluorophore properties such as photobleaching or other forms of photoswitching, which introduce a variance in photon emission, it is not trivial to choose optimal imaging parameters and to predict the spatial resolution. In this paper, we analytically derive a theoretical framework for estimating the achievable resolution of a microscope depending on parameters such as photoswitching, labeling densities, exposure time and sampling. We developed a numerical simulation software to analyze the impact of reversibly switchable probes in RESOLFT imaging.

Impact of Nanoparticle-Support Interactions in Co3O4/Al2O3 Catalysts for the Preferential Oxidation of Carbon Monoxide.

Different supporting procedures were followed to alter the nanoparticle-support interactions (NPSI) in two Co3O4/Al2O3 catalysts, prepared using the reverse micelle technique. The catalysts were tested in the dry preferential oxidation of carbon monoxide (CO-PrOx) while their phase stability was monitored using four complementary in situ techniques, viz., magnet-based characterization, PXRD, and combined XAS/DRIFTS, as well as quasi in situ XPS, respectively. The catalyst with weak NPSI achieved higher CO2 yields and selectivities at temperatures below 225 °C compared to the sample with strong NPSI. However, relatively high degrees of reduction of Co3O4 to metallic Co were reached between 250 and 350 °C for the same catalyst. The presence of metallic Co led to the undesired formation of CH4, reaching a yield of over 90% above 300 °C. The catalyst with strong NPSI formed very low amounts of metallic Co (less than 1%) and CH4 (yield of up to 20%) even at 350 °C. When the temperature was decreased from 350 to 50 °C under the reaction gas, both catalysts were slightly reoxidized and gradually regained their CO oxidation activity, while the formation of CH4 diminished. The present study shows a strong relationship between catalyst performance (i.e., activity and selectivity) and phase stability, both of which are affected by the strength of the NPSI. When using a metal oxide as the active CO-PrOx catalyst, it is important for it to have significant reduction resistance to avoid the formation of undesired products, e.g., CH4. However, the metal oxide should also be reducible (especially on the surface) to allow for a complete conversion of CO to CO2 via the Mars-van Krevelen mechanism.

Anticipating, measuring, and minimizing MEMS mirror scan error to improve laser scanning microscopy's speed and accuracy.

We describe a method to speed up microelectromechanical system (MEMS) mirror scanning by > 20x, while also improving scan accuracy. We use Landweber deconvolution to determine an input voltage which would produce a desired output, based on the measured MEMS impulse response. Since the MEMS is weakly nonlinear, the observed behavior deviates from expectations, and we iteratively improve our input to minimize this deviation. This allows customizable MEMS angle vs. time with <1% deviation from the desired scan pattern. We demonstrate our technique by optimizing a point scanning microscope's raster patterns to image mammal submandibular gland and pollen at ~10 frames/s.

Design Challenges of an Episode-Based Payment Model in Oncology: The Centers for Medicare & Medicaid Services Oncology Care Model.

The Centers for Medicare & Medicaid Services developed the Oncology Care Model as an episode-based payment model to encourage participating practitioners to provide higher-quality, better-coordinated care at a lower cost to the nearly three-quarter million fee-for-service Medicare beneficiaries with cancer who receive chemotherapy each year. Episode payment models can be complex. They combine into a single benchmark price all payments for services during an episode of illness, many of which may be delivered at different times by different providers in different locations. Policy and technical decisions include the definition of the episode, including its initiation, duration, and included services; the identification of beneficiaries included in the model; and beneficiary attribution to practitioners with overall responsibility for managing their care. In addition, the calculation and risk adjustment of benchmark episode prices for the bundle of services must reflect geographic cost variations and diverse patient populations, including varying disease subtypes, medical comorbidities, changes in standards of care over time, the adoption of expensive new drugs (especially in oncology), as well as diverse practice patterns. Other steps include timely monitoring and intervention as needed to avoid shifting the attribution of beneficiaries on the basis of their expected episode expenditures as well as to ensure the provision of necessary medical services and the development of a meaningful link to quality measurement and improvement through the episode-based payment methodology. The complex and diverse nature of oncology business relationships and the specific rules and requirements of Medicare payment systems for different types of providers intensify these issues. The Centers for Medicare & Medicaid Services believes that by sharing its approach to addressing these decisions and challenges, it may facilitate greater understanding of the model within the oncology community and provide insight to others considering the development of episode-based payment models in the commercial or government sectors.

Lithium and boron as interstitial palladium dopants for catalytic partial hydrogenation of acetylene.

It is demonstrated that light elements, including lithium and boron atoms, can take residence in the octahedral (interstitial) site of a Pd lattice by modifying the electronic properties of the metal nanoparticles, and hence the adsorptive strength of a reactant. The blocking of the sub-surface sites to H in the modified materials results in significantly higher selectivity for the partial catalytic hydrogenation of acetylene to ethylene.

PFG NMR and Bayesian analysis to characterise non-Newtonian fluids.

Many industrial flow processes are sensitive to changes in the rheological behaviour of process fluids, and there therefore exists a need for methods that provide online, or inline, rheological characterisation necessary for process control and optimisation over timescales of minutes or less. Nuclear magnetic resonance (NMR) offers a non-invasive technique for this application, without limitation on optical opacity. We present a Bayesian analysis approach using pulsed field gradient (PFG) NMR to enable estimation of the rheological parameters of Herschel-Bulkley fluids in a pipe flow geometry, characterised by a flow behaviour index n, yield stress τ0, and consistency factor k, by analysis of the signal in q-space. This approach eliminates the need for velocity image acquisition and expensive gradient hardware. We investigate the robustness of the proposed Bayesian NMR approach to noisy data and reduced sampling using simulated NMR data and show that even with a signal-to-noise ratio (SNR) of 100, only 16 points are required to be sampled to provide rheological parameters accurate to within 2% of the ground truth. Experimental validation is provided through an experimental case study on Carbopol 940 solutions (model Herschel-Bulkley fluids) using PFG NMR at a 1H resonance frequency of 85.2MHz; for SNR>1000, only 8 points are required to be sampled. This corresponds to a total acquisition time of <60s and represents an 88% reduction in acquisition time when compared to MR flow imaging. Comparison of the shear stress-shear rate relationship, quantified using Bayesian NMR, with non-Bayesian NMR methods demonstrates that the Bayesian NMR approach is in agreement with MR flow imaging to within the accuracy of the measurement. Furthermore, as we increase the concentration of Carbopol 940 we observe a change in rheological characteristics, probably due to shear history-dependent behaviour and the different geometries used. This behaviour highlights the need for online, or inline, rheological characterisation in industrial process applications.