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.

Keywords: Endothelial cells; FIB; Golgi apparatus; SBF-SEM; Weibel-Palade body.

Figure 1

Focused ion beam milling for serial block face (SBF)-scanning electron microscopy (SEM). Overview of the imaging setup and the sample. (A) Schematic representation showing the beam line of the electrons of the scanning electron microscope and the ions of the focused ion beam (FIB) with respect to the sample. The sample is tilted such that the electron beam can image from the top whereas the FIB can remove material from the side. A trench is created by the FIB to make the sample accessible for SBF imaging. (B) Low magnification overview imaged by the SEM showing the trench created for SBF-SEM imaging. Between the dashed lines, the monolayer of cell material is observed. Scale bar is 5 μm.

Figure 2

Organelles and subcellular structures by serial block face (SBF)-scanning electron microscopy (SEM). Overview of the subcellular structures and cell organelles in endothelial cells observed by SBF-SEM. (A) The extensive membrane stacks of the Golgi apparatus (Go). (B) Membrane network of the endoplasmic reticulum. In addition, also a lysosome (Ly) and parts of mitochondria (Mi) are observed. (C) Mitochondrion in which the internal cristae are clearly resolved (arrow). (D and D’) Pair of centrosomes. (D) Centrosome from the top. (D’) Centrosome from the side. (E) Microtubule (arrowheads) running just beneath the plasma membrane. (F) Caveolae at the plasma membrane. Scale bar panel (A) is 1 μm. Scale bar panel (B) and (C) is 500 nm. Scale bar panels (D)–(F) is 200 nm.

Figure 3

Weibel–Palade bodies imaged by SEM and TEM. Weibel–Palade bodies (WPBs) imaged by SEM and TEM in endothelial cells. (A) SBF-SEM slice showing cross-sectioned WPBs (arrows). (B) Longitudinal-sectioned WPB imaged by TEM displaying internal striations of Von Willebrand factor tubules. (C) Inverted SEM image of a longitudinal-sectioned WPB that displays a uniform stained interior. (D) Immature WPBs (arrows) near the Golgi apparatus (Go) imaged by TEM. Separate VWF tubules are clearly visible. (E) Inverted SEM image of an immature WPB near the Golgi apparatus (Go). Interior is less electron dense than the WPB in panel C but the VWF tubules are not resolved. Scale bar panel (A) is 1 μm. Scale bar panels (B)–(E) is 500 nm.

Figure 4

Serial block face (SBF)-scanning electron microscopy (SEM) reveals Weibel–Palade bodies in close association with the Golgi. Analysis and modelling of a large SBF-SEM stack to study WPBs in relation to the Golgi apparatus in endothelial cells. (A) Three-dimensional overview of the data set. The data set consist of almost 1600 images and was acquired at 25 000× magnification using a 10 nm slice thickness. For processing, the data were binned two times in x and y which resulted in a voxel resolution of 7.4 × 7.4 × 10 nm. (B) Segmentation of the volume reveals the large size of the Golgi apparatus (green) and the WPBs (red) that are in close association with the Golgi. In addition, we segmented the peripheral WPBs (yellow) and the nucleus (blue). The inserts show one of the WPBs that was found in close relationship with the Golgi. Scale bar is 500 nm. To have a better view on the ultrastructure, the data were rotated with respect to the orientation in which the data were acquired for panels (A) and (B). (C) Segmentation of the cell membrane additionally reveals the confined and flat morphology of the endothelial cell. Scale bar is 1 μm.