Misjaël N. Lebbink

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.

Focused ion beam‐scanning electron microscope: exploring large volumes of atherosclerotic tissue

Atherogenesis is a pathological condition in which changes in the ultrastructure and in the localization of proteins occur within the vasculature during all stages of the disease. To gain insight in those changes, high‐resolution imaging is necessary. Some of these changes will only be present in a small number of cells, positioned in a ‘sea’ of non‐affected cells. To localize this relatively small number of cells, there is a need to first navigate through a large area of the sample and subsequently zoom in onto the area of interest. This approach enables the study of specific cells within their in vivo environment and enables the study of (possible) interactions of these cells with their surrounding cells/environment. The study of a sample in a correlative way using light and electron microscopy is a promising approach to achieve this; however, it is very laborious and additional ultrastructural techniques might be very valuable to find the places of interest.

Architecture-Dependent Distribution of Mesopores in Steamed Zeolite Crystals as Visualized by FIB-SEM Tomography†

Break on through: Steaming-induced mesopores of individual ZSM-5 crystals were studied by a combination of focused ion beam (FIB) and scanning electron microscopy (SEM) tomography (see picture). In this manner, quantitative insight into the width, length, morphology, and distribution of mesopores generated within zeolite crystals has been obtained. Keywords:crystal intergrowth;scanning probe microscopy;mesoporosity;tomography;zeolites

Spiral Coating of the Endothelial Caveolar Membranes as Revealed by Electron Tomography and Template Matching

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3‐D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high‐pressure frozen, freeze‐substituted and epon‐embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises – as confirmed by 3‐D immunolabeling for caveolin of ‘intact’ cells –bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi‐caveolar bodies. Application of template matching to tomograms allowed the 3‐D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze‐etched caveolae from fibroblasts. Meticulous 3‐D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the ‘intact’ caveolar system, setting a detailed morphological basis for its functional diversity.

FIB-SEM cathodoluminescence tomography: practical and theoretical considerations

Focused ion beam–scanning electron microscope (FIB‐SEM) tomography is a powerful application in obtaining three‐dimensional (3D) information. The FIB creates a cross section and subsequently removes thin slices. The SEM takes images using secondary or backscattered electrons, or maps every slice using X‐rays and/or electron backscatter diffraction patterns. The objective of this study is to assess the possibilities of combining FIB‐SEM tomography with cathodoluminescence (CL) imaging. The intensity of CL emission is related to variations in defect or impurity concentrations. A potential problem with FIB‐SEM CL tomography is that ion milling may change the defect state of the material and the CL emission. In addition the conventional tilted sample geometry used in FIB‐SEM tomography is not compatible with conventional CL detectors.

D-CAT: Density and Clustering Annotation Tool for three dimensional electron microscopic volumes.

Three dimensional (3D) electron microscopy techniques have become valuable tools for investigating cellular architecture and the processes that govern it. A vast amount of information is available in every 3D tomogram but the options for presenting this information in a clear and visually appealing way are limited. To address this, we developed D-CAT; a MatLab-application to accurately visualize the distribution of membrane proteins and/or membrane-bound structures. Presence (density) and distribution (clustering, depletion) are presented as color-coded areas on membranes. By using IMOD models both as input and output format, we ensure that the application fits within workflows common in the field of 3D electron microscopy.

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.

Electron tomography & template matching of biological membranes

In recent years, electron tomography has proven to be a valuable addition to existing EM techniques, providing new and exiting insights into cellular architecture in three dimensions. Constant technical developments have played a crucial role in establishing the role of the technique in modern electron microscopic studies. The next technical bottleneck to overcome is that of a reliable, objective, and (semi)-automatic analysation of the acquired data. One such approaches is three dimensional template matching based on normalized cross-correlation analysis (figure 1) [1].