Quantitative Analysis of Immature Secretory Granules in Beta Cells of Mouse Pancreatic Islets by Serial Block-Face Scanning Electron Microscopy

Abstract

Serial block-face scanning electron microscopy (SBF-SEM) can provide high-resolution structural information from large volumes of biological tissue at the nanometer scale. By using SBF-SEM with a ∼1 kV electron probe that is scanned across the surface of a plastic embedded block of tissue, it is possible to image tissue volumes as large as 100,000 cubic micrometers with 5 nanometer voxel size in the plane of the block face and 25 nanometer normal to the block face. A sensitive detector collects backscattered electrons from heavy atoms staining ultrastructure near the surface of the block, and a microtome built in to the specimen stage cuts a thin layer from block face to expose a surface for imaging. Repetition of these steps produces an image stack that can be aligned, analyzed, and segmented. We have used SBF-SEM to study immature secretory granules in beta cells of mouse islets. Secretory granules contain insulin that is released in response to elevated blood glucose levels. Our objective was to use granule morphology to define their state of maturity. Immature granules contain proinsulin, and have lightly stained cores surrounded by thin halos, in contrast to mature granules that have more densely stained insulin cores surrounded by wide halos. From the SBF-SEM image stacks, we identified mature and immature secretory granules and measured the ratio of core volume to total granule volume. We also measured the relative abundances of immature granules and mature granules, which enabled us to estimate the maturation time for the beta granules by considering the measured release times of isotopically labeled insulin.