We have applied serial block-face scanning electron microscopy (SBF-SEM) using a Zeiss SIGMA-VP SEM and a Gatan 3View system to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs about 200 to 300 micrometers in size, which secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9.5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections. The spatial resolution of SBF-SEM normal to the block face is currently limited to approximately 25 nanometers by the minimum slice thickness that can be removed using the ultramicrotome that is built into the SEM's specimen stage. We have carried out Monte Carlo simulations of electron trajectories within the block face to determine whether it is possible to obtain sub-25 nanometer z-resolution by recording backscattered images at different beam energies to probe different sub-surface depths within the block. Results show the feasibility of achieving a z-resolution of around 5 nanometers by combining backscattered images at 1.4 keV and 6.8 keV. Experiments are in progress to test this capability on well-defined test specimens.
| He, Q; Hsueh, M; Zhang, G et al. (2018) Biological serial block face scanning electron microscopy at improved z-resolution based on Monte Carlo model. Sci Rep 8:12985 |
| McBride, E L; Rao, A; Zhang, G et al. (2018) Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography. J Struct Biol 202:216-228 |
| Zhu, Lu; Almaça, Joana; Dadi, Prasanna K et al. (2017) ?-arrestin-2 is an essential regulator of pancreatic ?-cell function under physiological and pathophysiological conditions. Nat Commun 8:14295 |
| Shomorony, A; Pfeifer, C R; Aronova, M A et al. (2015) Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells. J Microsc 259:155-164 |
| Pfeifer, C R; Shomorony, A; Aronova, M A et al. (2015) Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM. J Struct Biol 189:44-52 |
