In this paper the possibilities of focused ion beam (FIB) applications in microsystem technology are reviewed. After an introduction to the technology and the operating principles of FIB, two classes of applications are described. First the subject of FIB for microsystem technology inspection, metrology and failure analysis is outlined. A procedure for cross sectioning on samples is presented, as well as some examples of how this technique can be applied to study processing results. The second part of the paper is on the use of FIB as a tool for maskless micromachining. Both subtractive (etching) and additive (deposition) techniques are discussed, as well as the combination of FIB implantation of silicon with subsequent wet etching. We will show the possibility to fabricate three-dimensional structures on a micrometre scale, and give examples of recent realizations thereof.

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.O ver 40 million people worldwide are blind. Macular degeneration accounts for Ϸ8 million cases of blindness. Although the cause of macular degeneration is not known, 1 potential treatment is cell therapy. Stem cells hold great promise for regenerative medicine, and recently many efforts have been devoted to finding suitable candidates for retinal transplants. These candidates include photoreceptor progenitors (1) and embryonic stem cells (2, 3). Others have looked to the ciliary epithelium (CE) as a potential source of retinal stem cells (4, 5).In 2000, Tropepe and colleagues revealed that the CE of the mouse eye can be dissociated, maintained in culture at clonal density and stimulated to form clonogenic spheres (5). The CEderived spheres were pigmented, expressed nestin, and could be dissociated and replated to form spheres up to 8 times. When transferred to differentiation conditions in culture, cells from the CE-derived spheres up-regulated genes found in rod photoreceptors, bipolar neurons, and Müller glia (5). These findings were later extended to CE isolated from postmortem human eyes (4). Based on these and other data, it was suggested that the sphere-forming cells in the mammalian CE are retinal stem cells (RSCs) and, thus, hold promise for therapeutic replacement of retinal neurons lost to disease or degeneration.Given the potential impact of cell replacement ...

In this work, some of the possibilities of focused ion beams for applications in microsystem technology are explored. Unlike most previous studies, the emphasis is on `additive' techniques, i.e. localized maskless deposition of metals and insulators. More precisely, we will show the possibility of fabricating small three-dimensional structures, using focused ion beam deposition of silicon oxide. Deposition examples will show that the technique is most promising for small post-processing steps or prototyping, because of its high degree of flexibility. Furthermore, an investigation into the mechanical properties of the deposited material is presented. More specifically, the Young's modulus of the deposited silicon oxide is determined.

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.

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