Amir Avishai

Myelination and Axonal Electrical Activity Modulate the Distribution and Motility of Mitochondria at CNS Nodes of Ranvier

Energy production presents a formidable challenge to axons as their mitochondria are synthesized and degraded in neuronal cell bodies. To meet the energy demands of nerve conduction, small mitochondria are transported to and enriched at mitochondrial stationary sites located throughout the axon. In this study, we investigated whether size and motility of mitochondria in small myelinated CNS axons are differentially regulated at nodes, and whether mitochondrial distribution and motility are modulated by axonal electrical activity. The size/volume of mitochondrial stationary sites was significantly larger in juxtaparanodal/internodal axoplasm than in nodal/paranodal axoplasm. With three-dimensional electron microscopy, we observed that axonal mitochondrial stationary sites were composed of multiple mitochondria of varying length, except at nodes where mitochondria were uniformly short and frequently absent altogether. Mitochondrial transport speed was significantly reduced in nodal axoplasm compared with internodal axoplasm. Increased axonal electrical activity decreased mitochondrial transport and increased the size of mitochondrial stationary sites in nodal/paranodal axoplasm. Decreased axonal electrical activity had the opposite effect. In cerebellar axons of the myelin-deficient rat, which contain voltage-gated Na+ channel clusters but lack paranodal specializations, axonal mitochondrial motility and stationary site size were similar at Na+ channel clusters and other axonal regions. These results demonstrate juxtaparanodal/internodal enrichment of stationary mitochondria and neuronal activity-dependent dynamic modulation of mitochondrial distribution and transport in nodal axoplasm. In addition, the modulation of mitochondrial distribution and motility requires oligodendrocyte–axon interactions at paranodal specializations.

Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology

Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl⁻/⁻) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field.

Focused ion beam (FIB) nanomachining of silicon carbide (SiC) stencil masks for nanoscale patterning

We report on experimental explorations of using focused ion beam (FIB) nanomachining of different types of silicon carbide (SiC) thin membranes, for making robust, high-quality stencil masks for new emerging options of nanoscale patterning. Using thin films and membranes in polycrystalline SiC (poly-SiC), 3C-SiC, and amorphous SiC (a-SiC) with thicknesses in the range of t~250nm−1.6μm, we have prototyped a series of stencil masks, with nanoscale features routinely down to ~100nm.

Transmission Electron Diffraction Investigation of White Etching Areas in Bearing Steels: A Comparison Between TKD and TEM

White etched areas (WEAs) are microstructural alternations in bearings induced by dynamic loading conditions. As indicated by the name, WEAs are more resistance to etching by chemicals and demonstrate “white” contrast under the optical microscope (Figure 1). The occurrence of WEAs can lead to the initiation of cracks, which can propagate and cause premature failures of bearing components. [1] Our previous investigations using transmission electron microscopy (TEM) [2] showed that the WEAs are very fine-grained (10-100 nm in diameter) severely plastically deformed zones. The grains in the WEAs are much finer than those in the original microstructure. Microcracks and voids were also observed in the WEAs, and pre-existing carbides are no longer visible. We emphasize that one of the major challenges working with WEAs is the high level of plastic deformation combined with a very fine-grained microstructure.

Orientation Mapping by Precession Transmission Electron Microscopy

Precession transmission electron microscopy (PTEM) is currently a very “hot” topic [1]. One of its major applications is orientation mapping. While conventional electron backscattered diffraction (EBSD) and transmission Kikuchi diffraction (TKD) [2] rely on Kikuchi pattern analysis, PTEM (ASTAR system by Nanomegas) acquires micro-diffraction patterns for orientation information. Therefore, even though both methods work in most samples, it is possible that for certain samples PTEM may have an advantage over the EBSD/TKD technique.

Characterization of Dental Bonded Interface Degradation Using Focused Ion Beam and High-Resolution Transmission Electron Microscopy

Esthetic bonded restorations are the most popular category of dental restoration. In order to promote adequate bond strengths, resin monomers must interlock micromechanically with the exposed collagen network of dental structures. Human dentin must be etched or modified by the use of etchants or acidic monomers, before the application of resin monomers. Microchannels are created by the action of the etchants, and the monomers must flow into these channels of nanometric dimensions forming a polymer and collagen network called “hybrid layer” [1]. Remaining water in the collagen network can dilute or modify the monomer, reducing the ability to entirely fill and seal the etched surface. Therefore, porosity within the hybrid layer poses a problem, because of retained water [2]. To elucidate the characteristics of the resin-impregnated layer, long-term investigation is needed.

Are Axonal Mitochondria Really Enriched at Nodes of Ranvier? A Three Dimensional Serial Ion Ablation SEM (FIB/SEM) Study.

To meet the energy demands of nerve conduction, small mitochondria are transported from the neuronal cell body and enriched at mitochondrial stationary sites located throughout the axon. Determining whether and how myelination locally regulates mitochondrial structure and function in axons is fundamental to understanding axonal biology and pathology [1]. Nodes of Ranvier are the sites of action potential propagation, and previous EM studies of myelinated axons have suggested that axons are enriched in nodal axonal cytoplasm (axoplasm). Paradoxically, several studies noted that mitochondria were frequently absent at nodes of Ranvier [2,3]. Numbers of nodes with and without mitochondria had not been quantified, presumably to difficulties in obtaining extensive 3D EM datasets including entire nodes.

Serial Sectioning in SEM: Challenges and Opportunities

*Dept. of Mat. Sci. and Eng., Case Western Reserve University, Cleveland, OH, 44106. **Dept. of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44120. ***Dept. of Molecular Bio. & Microbiology, CWRU School of Medicine, Cleveland, OH, 44106. ****Dept. of Restorative Sciences, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, 90089. *****Dept. of Pharmacology, Case Western Reserve University, Cleveland, OH, 44106. ******Cell and Tissue Imaging Center, St. Jude Children’s Research Hospital, Memphis, TN 38105.