Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

New methods for the study and fabrication of nano-structured materials using FIB SEM.

Techniques for characterisation and methods for fabrication at the nanoscale are becoming more powerful, giving new insights into the spatial relationships between nanostructures and greater control over their development. A case in point is the application of state-of-the-art focused ion beam technology (FIB), in combination with high-performance scanning electron microscopy (SEM), to generate cross-sections into bulk material and create a sequential image series. These two-dimensional images can then be correlated and rendered into a three-dimensional representation. In addition, site-specific, ultra-thin lamellar specimens can be made for observation in the transmission electron microscope (TEM) or scanning transmission electron microscope (STEM), with the further advantage that FIB cutting through hard-soft interfaces poses fewer difficulties compared to ultramicrotomy. Another big impact of FIB SEM on nanotechnology is the ability to use either ions or electrons to perform advanced nanolithography, via etching or chemical vapour deposition. In all cases, numerous parameters must be considered in order to achieve high quality results, particularly where stringent critical dimensions are required or when dealing with challenges such as electrically insulating and/or soft materials. We have developed strategies to address these issues, enabling results across a wide range of nanotechnology applications.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Controlling the electrodeposition of mesoporous metals for nanoplasmonics.

We have investigated the mechanism for formation of large-scale ordered structures of copper and silver by direct electrodeposition through colloidal crystal templates. It is demonstrated that the morphology is controlled by the diffusion of reactant species (metal cations and oxygen molecules) from the bulk solution to the electrode surface. The Cu system can be tuned to fill or not to fill the original drying cracks in the template in order to produce isolated or interconnected domains. This has great potential for the formation of individual or interconnected antennae in nanoplasmonic structures.