DOI: 10.25378/janelia.13114499.v1
title: Isotropic 3D electron microscopy reference data of isolated murine pancreatic islets treated with high glucose (jrc_mus-pancreas-1)

description: Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structure with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Here, we overcome these limitations by long-term imaging whole cells and tissues via the enhanced Focus Ion Beam Scanning Electron Microscopy (FIB-SEM) platform in high resolution mode with month-long acquisition duration. We use this approach to generate reference 3D image data sets at 4-nm isotropic voxels. Together with subsequent segmentation, we hope to create a reference library to explore comprehensive quantification of whole cells and all their constituents, thus addressing questions related to cell identities, cell morphologies, cell-cell interactions, as well as intracellular organelle organization and structure.<b>Pancreatic islets </b>are micro-organs consisting mainly of beta, alpha, delta, polypeptide cells and endothelial cells. Beta cells are the majority of the islet cells. They secrete insulin, which is stored in secretory granules (SGs), in order to maintain blood glucose homeostasis. Beta cells within the islet are heterogeneous in their response to glucose. Furthermore, not all insulin SGs within beta cells have the same likelihood of being released. Large-scale high-resolution FIB-SEM enables biologists to investigate the ultrastructural differences between beta cells within an islet as well as features that require higher resolution such as ribosomes and the cytoskeleton. Here we present a 200-GB volume with several complete beta cells that are entirely included within the 30 x 20 x 25 µm<sup>3</sup> volume. The large field of view allows to image the interaction of beta cells, e.g. full primary cilia and their connections to neighboring cells and the intermingling of microvilli. At 4-nm voxel sampling, we observed ultrastructural differences between insulin SGs. For the first time, we report close contacts of ER and insulin SGs in 3D. Ultimately, such a resolution allows the sharp definition of microtubules, single ribosomes and Golgi stacks for quantitative measurements. The seamless imaging resulting in precisely aligned stacks furthermore facilitates automated 3D segmentation approaches, which allows for new biological insights. As such, it unveils a comprehensive 3D representation of microtubule networks and how they interact with other organelles, as well as microtubule remodeling as a result of glucose stimulation.Sample: Wild-type mouse pancreatic islets treated with high glucose.<br>Protocol: High-pressure freezing and freeze-substitution with 2% OsO4, 1% UA, 0.5% GA, 5% H2O in acetone with 1% methanol, followed by 0.2% TCH in 80% methanol at RT, 2% OsO4 in acetone at RT, 1% UA in acetone + 10% methanol.Contributions: Sample provided by Andreas Mueller and Michele Solimena (Paul Langerhans Institute, Dresden), prepared for imaging by Song Pang (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).Dataset ID: jrc_mus-pancreas-1Final voxel size (nm): 4.00 x 4.00 x 3.40 (X, Y, Z)Dimensions (µm): 30 x 20 x 25 (X, Y, Z)Acquisition date: 2019-03-01Dataset URL: s3://janelia-cosem-datasets/jrc_mus-pancreas-1/jrc_mus-pancreas-1.zarr/recon-1/em/Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-pancreas-1<br>Publication: “Three-dimensional FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse beta cells” by A. Müller et al. bioRxiv 2020.10.07.329268. (2020); “Isotropic 3D electron microscopy reference library of whole cells and tissues” by C. Shan Xu et al. (in preparation)