title: Towards the imaging of Weibel-Palade body biogenesis by serial block face-scanning electron microscopy.
abstract: Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face-scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three-dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre-scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three-dimensional analysis. Here, we explore focused ion beam facilitated serial block face-scanning electron microscopy to study the endothelial cell-specific storage organelles, the Weibel-Palade bodies, during their biogenesis at the Golgi apparatus. Weibel-Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face-scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel-Palade bodies and Golgi stacks which became conceivable in large-scale three-dimensional data. We demonstrate that serial block face-scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors: M J
authors: MJ
authors: Mourik
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors: F G A
authors: FGA
authors: Faas
authors: Carl Zeiss Microscopy GmbH, Munich, Germany.
authors: H
authors: H
authors: Zimmermann
authors: Department of Thrombosis and Hemostasis, Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands.
authors: J
authors: J
authors: Eikenboom
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors: A J
authors: AJ
authors: Koster
mesh_terms: D002478:Cells, Cultured; D055032:Electron Microscope Tomography / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology; D006056:Golgi Apparatus / Q000648:ultrastructure; D016591:Histocytological Preparation Techniques / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008867:Microtomy / Q000379:methods; D001678:Organelle Biogenesis; D016610:Tissue Embedding; D020302:Weibel-Palade Bodies / Q000502:physiology / Q000648:ultrastructure*
keywords: Endothelial cells; FIB; Golgi apparatus; SBF-SEM; Weibel-Palade body
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