Robyn Webb

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.

The composition of interfacial material from skim milk foams.

The foaming properties of skim milk vary with temperature of foaming in the range from 5 to 85°C, with foams of maximum stability being formed at approximately 45°C. This paper reports the significance of different milk fractions in the foam and concludes that the micellar casein fraction plays an important role in stabilization of milk foam formed at higher temperatures. This finding was supported by the fact that added calcium chloride increased and calcium-chelating agents decreased foam stability. These effects were attributed to the increase and decrease, respectively, in the amount of micellar casein in the milk. Furthermore, bubble ghost material sedimented by low-speed centrifugation of foam was found to contain predominantly caseins, and electron micrographs of foams formed at 45°C clearly showed casein micelles spread over the interface. However, other structures observed in the electron micrographs suggest that soluble milk proteins and possibly polar lipids are also present in the foams and play a role in formation of milk foams.

Cell polarity defines three distinct domains in pancreatic beta cells

ABSTRACT The structural organisation of pancreatic β-cells in the islets of Langerhans is relatively unknown. Here, using three-dimensional (3D) two-photon, 3D confocal and 3D block-face serial electron microscopy, we demonstrate a consistent in situ polarisation of β-cells and define three distinct cell surface domains. An apical domain located at the vascular apogee of β-cells, defined by the location of PAR-3 (also known as PARD3) and ZO-1 (also known as TJP1), delineates an extracellular space into which adjacent β-cells project their primary cilia. A separate lateral domain, is enriched in scribble and Dlg, and colocalises with E-cadherin and GLUT2 (also known as SLC2A2). Finally, a distinct basal domain, where the β-cells contact the islet vasculature, is enriched in synaptic scaffold proteins such as liprin. This 3D analysis of β-cells within intact islets, and the definition of distinct domains, provides new insights into understanding β-cell structure and function. Summary: 3D imaging methods identify three structural and functional domains within β-cells in islets: apical, lateral and basal.

Integration of observational and analytical methodologies to characterize organic matter in early archaean Rocks: Distinguishing biological from abiotically synthesized carbonaceous matter structures

Transmission Electron Microscopy (TEM) was applied to observe and characterize carbonaceous materials (CM) extracted from black cherts and argillite in drill core from the Warrawoona Group of the Pilbara Craton, Western Australia. The black chert came from a ‘white smoker type’ seafloor deposit in the ca. 3.49 Ga Dresser Formation, whereas the black argillites were obtained from the 3.46 Ga Apex Basalt. The samples were observed and analyzed in TEM combined with electron dispersive spectral analysis (EDS), high resolution TEM (HRTEM) to determine molecular ordering, and C-isotope geochemistry. The TEM and HRTEM observations revealed significant morphological and structural differences between the carbonaceous materials of the Dresser and Apex samples enabling interpretations in terms of primary and secondary origins, as well as metamorphic history.

Journey to the centre of the cell: Virtual reality immersion into scientific data

Visualization of scientific data is crucial not only for scientific discovery but also to communicate science and medicine to both experts and a general audience. Until recently, we have been limited to visualizing the three‐dimensional (3D) world of biology in 2 dimensions. Renderings of 3D cells are still traditionally displayed using two‐dimensional (2D) media, such as on a computer screen or paper. However, the advent of consumer grade virtual reality (VR) headsets such as Oculus Rift and HTC Vive means it is now possible to visualize and interact with scientific data in a 3D virtual world. In addition, new microscopic methods provide an unprecedented opportunity to obtain new 3D data sets. In this perspective article, we highlight how we have used cutting edge imaging techniques to build a 3D virtual model of a cell from serial block‐face scanning electron microscope (SBEM) imaging data. This model allows scientists, students and members of the public to explore and interact with a “real” cell. Early testing of this immersive environment indicates a significant improvement in students’ understanding of cellular processes and points to a new future of learning and public engagement. In addition, we speculate that VR can become a new tool for researchers studying cellular architecture and processes by populating VR models with molecular data.

Vapor-Phase Hydrothermal Growth of Novel Segmentally Configured Nanotubular Crystal Structure

A new form of nanotubular crystal structure is directly grown by a vapor-phase hydrothermal method via an epitaxial orientated crystal growth mechanism. The as-prepared nanotubes possess a unique multi-tunnel core-shell layered nanotubular structure with droplet shaped polygonal periphery and segmental crystal configuration. They are dimension-tunable and demonstrate superior ion exchange properties in terms of exchange rate and ion accommodating capacity.

Quick Freeze Substitution Processing of Biological Samples for Serial Block-face Scanning Electron Microscopy

Until recently the only way to obtain three-dimensional electron microscopy data over a large area of a biological sample was via serial sectioning, a tedious and technically challenging technique. Serial block-face scanning electron microscopy (SBF-SEM) has recently been used as a technique to obtain similar results to serial sectioning transmission electron microscopy but in a simpler and semi-automated way. It involves working with a resin embedded sample similar to that used for transmission electron microscopy. However, the imaging is performed on the block-face rather than the section cut from it. Image collection is done using a backscattered electron detector in a scanning electron microscope. An image is taken of the block-face and then a thin section is cut from the block using a diamond knife. The newly exposed block-face is then imaged, another section cut from it, it is imaged again etc. This process of imaging and sectioning is performed over and over building up a three-dimensional data set. Two commercial instruments are available that will perform SBF-SEM: the Gatan 3View and the FEI Teneo Volumescope.