D Wall

Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling

### Introduction Analyzing the synaptic basis of neuronal circuits within a volume of brain tissue requires electron microscopy. With a resolution capable of seeing the smallest synaptic contacts, this method uses different sectioning techniques to produce serial images suitable for seeing the

Electron Tomography on Micrometer-Thick Specimens with Nanometer Resolution

Transmission electron microscopy (TEM) is a well-established technique to explore matter down to the atomic scale. TEM tomography methods have been developed to obtain volume information at the mesoscopic dimensions of devices or complex mixtures of multiphase objects with nanometer resolution, but these methods are in general only applicable to relatively thin specimens with a few hundred nanometer thickness at most. Here we introduce an approach based on scanning TEM (STEM) tomography that pushes the resolution in three dimensions down to a few nanometers for several micrometer ultrathick specimens using a conventional TEM with 300 kV accelerating voltage, and we demonstrate its versatility for materials research and nanotechnology.

Chemical speciation of nanoparticles surrounding metal-on-metal hips

Spectromicroscopy of tissue surrounding failed CoCr metal-on-metal hip replacements detected corroded nanoscale debris in periprosthetic tissue in two chemical states, with concomitant mitochondrial damage. The majority of debris contained Cr(3+), with trace amounts of oxidised cobalt. A minority phase containing a core of metallic chromium and cobalt was also observed.

Birbeck Granule-Like “Organized Smooth Endoplasmic Reticulum” Resulting from the Expression of a Cytoplasmic YFP-Tagged Langerin

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of “Organized Smooth Endoplasmic Reticulum” (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a “double-lock” mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.

Tomographic imaging ultra-thick specimens with nanometer resolution

Transmission electron microscopy (TEM) is a well established and powerful technique to explore matter down to the atomic scale in two dimensions. Since real world objects have a three dimensional character the need to obtain volume information on nanometer scale is increasing especially on the mesoscopic dimensions of devices or complex mixtures of multiphase objects. TEM and scanning TEM (STEM) tomography methods have been developed in the past to fulfil this need, but these methods are limited to relatively thin specimens. In TEM this is caused mainly by the chromatic aberration and the dramatic increase of inelastic scattering in the sample, which leads ultimately to a strong resolution loss. Only high acceleration voltage TEM (above 1 million volts) allows for investigation of thicker specimens; this technique is, however, extremely expensive, far from being routine and highly destructive, in particular for organic matter. STEM tomography can minimize the effect of chromatic aberration, but is limited by the focal depth when large convergence angles are used. In this paper we establish a novel method based on dark field STEM tomography that pushes the resolution in all three dimensions down to a few nanometers for ultra-thick specimens of several micrometers at 300kV acceleration voltage.

Optimized Electron Column and Detection Scheme for Advanced Imaging and Analysis of Metals

In recent years, there has been a large growth in the development of detectors for FIB/SEM systems as the number of applications and techniques is growing. In-lens detectors have traditionally been optimized for imaging low take-off-angle back-scattered electrons with higher energies (low loss). This signal typically generates images which are high in materials contrast [1]. Conversely, below-lens detectors have the ability to capture much higher take-off-angle backscattered electrons due to their geometry [2]. Recent advances in detector segmentation with concentric rings have further allowed the separation of take-off angle signal without the need to change the working distance between the final lens and the sample. This higher take off angle delivers a strong channeling contrast in the specimen, while the highest take off angle reveals topographic information.

A Novel Automated Approach to Serial Block Face DualBeam Electron Microscopy for the Exploration of Cortical Circuits

The mammalian brain comprises interconnected neurons that communicate via synapses to form a bewilderingly complex network. In every micro-liter of cortical grey matter there are approximately one billion synapses arranged along several kilometers of axons. Visualizing these circuits and all their connections is paramount to being able to understand how the brain works. To achieve this would require an imaging technique that has both the resolution to distinguish the different types of synaptic connections as well as a wide enough field of view to identify all of them.