Ben Lich

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

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low‐kV BSE imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three‐dimensional micro‐ and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block‐face imaging using low‐kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)‐coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high‐vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

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.

Analysis of acute brain slices by electron microscopy: A correlative light–electron microscopy workflow based on Tokuyasu cryo-sectioning

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasus protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

High-Throughput Large Volume SEM Workflow using Sparse Scanning and In-painting Algorithms Inspired by Compressive Sensing

We are presenting a new extension to our Cell and Tissue/Neurobiology large volume imaging workflow, with the goal of increasing acquisition speed by more than five times. Instead of scanning dense square-grid frames, in the conventional way, our approach is here to explore the use of sparse scanning and inpainting techniques inspired by Compressive Sensing (CS) [1]. Sparse samples are obtained by pseudo-random scan patterns, and reconstruction algorithms are used to recover the dense volume data. The goal is to recover 3D datasets with minimum loss of information. Techniques inspired by CS gained wide attention over the last decade and are now being used in various applications where sensor bandwidth is a limiting factor. They have been recently explored for SEM and STEM applications [2][3]. In the context of nano-scale cell biology volume acquisition, we expect these techniques to ultimately increase the imaging throughput by nearly an order of magnitude. We will discuss additional advantages of this approach, such as the low-dose imaging of sensitive specimens, and the good compatibility with backscatter electron imaging. A key enabler of any sparse scan application to EM is the accurate control of scan locations. It has been shown in [2] and in our own experiments that precise positioning of the beam at the planned sampling locations is essential for a good CS reconstruction. We have developed advanced minimum-path scanning strategies to address this issue. The scanning technique is illustrated in Fig. 1, where the left two images show a conventional raster scan at 300ns dwell visiting a random set of points with the compressive sensing reconstruction obtained from such scan strategy. The right two images of Fig. 1 show an example minimum-path scan pattern and a much improved reconstruction result from images acquired with this second method. In future work we will compare pseudo-random sparse sampling, in combination with a reconstruction algorithm based on CS-inspired in-painting, to conventional grid sampling of the same effective dose, in combination with a de-noising algorithm, also based on CS. CS machine learning algorithms build patch-dictionaries, which are used as the building blocks for data representation [3]. During live acquisition runs, such dictionaries can be used to in-paint with high fidelity, the sparsely sampled datasets (Figure 2). We are implementing the new sparse scanning modules on SEM platforms, which also employ the Multi Energy Deconvolution SEM (MED-SEM) technology and Serial Block Face (SBF) imaging [4]. By incorporating CS, we will have an instrument allowing for both high-resolution isotropic imaging, and the fast acquisition of very large datasets (Figure 3).

Tomography of Biological Materials using Focused Ion Beam Sectioning and Backscattered Electron Imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional microand nanostructures. It has been widely applied in studying three-dimensional nanoand microstructures of materials such as metals and semi-conductors (e.g., Inkson et al., 2001; McGrouther and Munroe, 2007). In biology the method is extremely useful to study cell / implant interactions (Giannuzzi et al., 2007; Greve et al., 2007; Nalla et al., 2005). The density difference between the biological matter and the implant is so extreme that standard microtomy is very difficult if not impossible. An other application is to dig into biological objects and reveal the structures of interest (Drobne et al., 2007; Drobne et al., 2005a; Drobne et al., 2005b). The real power of FIB-SEM tomography, however, is to find the place of interest at low magnification in the SEM and then analyse the spot at higher magnification with the Slice and ViewTM (FEI Company) method, which means cutting a slice off with the ion beam and imaging the fresh surface with the electron beam, usually with the backscatter electron mode (De Winter et al., 2009; Heymann et al., 2006; Knott et al., 2008). More advanced investigations aim at using the focused ion beam scanning microscope as an ultramicrotome to create thick cryo-sections (Marko et al., 2006; Marko et al., 2007). In this presentation we will share our experience in analysing endothelial cells and atherosclerotic plaques of Epon embedded samples with FIBSEM tomography.

Focused Ion Beam Tomography of Insulating Biological and Geological Materials

The analysis of large volumes at high resolution gains more and more importance. Thanks to transmission electron tomography is has become obvious that a twodimensional representation can lead to incomplete interpretation and understanding of three-dimensional structures. Especially in cell biology new insight has been gained in the connection of intracellular organelles and their biogenesis [1–3].

Advances in 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 microliter of cortical grey matter there is 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. Synapses are specialized junctions whose sizes range from over a micron in diameter to less than 100nm. Their size means that they can only be identified using the electron microscope, which also allows them to be classified according to their function, ie. inhibitory or excitatory. However, to understand the connectivity between neurons in the cortex, large volumes of neural tissue needs to be imaged at ‘synaptic resolution’ and in three dimensions (3d). Traditionally serial section transmission electron microscopy has been the only method available in which images are acquired from sections that are cut and mounted on grids. These image representing a 3d volume can be used to reconstruct and analyze the structure of the elements within. This method is very labor intensive, requiring a greater deal of manual dexterity, and when small mistakes occur, a continuous dataset is lost. Recent studies have explored an alternative method for serial image acquisition in which the block face of resin-embedded neural tissue was imaged within a FEG SEM [1]. Sections were removed from the imaged face in the microscope using an ultra microtome and then immediately imaged by the SEM. This provides a series of aligned images of the tissue in the block face and has the clear advantage that image acquisition can be fully automated. However the quality of the sectioning is critically dependent on homogenous resin hardness which is difficult to maintain when the electron beam is being used at higher resolution and part of the block suffers from the differential heating effects.

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.