DOI: 10.25378/janelia.13114211.v2
title: Isotropic 3D electron microscopy reference data of wild-type, interphase HeLa cell (jrc_hela-2)

description: <b>This acquisition is part of the CellMap 2024 Segmentation Challenge</b><b>Challenge DOI:</b> https://doi.org/10.25378/janelia.c.7456966<b>Challenge Website:</b> https://cellmapchallenge.janelia.org/<b>Sample</b>: Wild-type, interphase HeLa cell<b>Sample Description</b>: 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.<br><b>HeLa cells</b>, human cervical cancer cells that are the oldest and most commonly used cell line, are easily cultured and widely used in cell biology labs as a basic model to test diverse hypotheses. Having such cells imaged in their entirety can provide a reference to which perturbations in growth, genetic, environment, etc. can be compared. Here we present a typical 3D data set on the example of the entire HeLa cell. The mitochondrial network is clearly identified, as well as 2D cross-sections of standard cellular organelles, such as centrosome, Golgi apparatus, and nuclear envelope. Notably, every example illustrates the advantages of isotropic 3D imaging for cell biology. No single 2D cross-section allows visualizing all centriole sub-distal appendages, however quick segmentation of 3D data set characterizes them easily. Stereotypical 2D images of the Golgi stacks do not reveal the fenestration details and long thin tubular extensions, that can only be seen in 3D. Polyribosomal chains on the nuclear envelope are mostly hidden in 2D cross-sections but easily resolved and detailed in 3D. The unique ability of enhanced FIB-SEM to image whole cells and tissues at 4-nm isotropic voxels over large volumes makes it an ideal tool to map <i>in toto</i> the 3D ultrastructural relationship in living systems.<br><b>Protocol</b>: High pressure freezing, freeze-substitution resin embedding with 2% OsO4 0.1% UA 3% H2O in acetone; resin embedding in Eponate 12.<b>Contributions</b>: Sample provided by Aubrey Weigel (HHMI/Janelia), prepared for imaging by Gleb Shtengel (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).<b>Acquisition ID</b>: jrc_hela-2<b>Final voxel size (nm)</b>: 4.0 x 4.0 x 5.24 (X, Y, Z)<b>Data dimensions (µm)</b>: 48 x 6 x 33 (X, Y, Z)<b>Imaging start date</b>: 2017-06-21<b>Imaging duration (days)</b>: 22<b>Primary energy (eV)</b>: 900<b>Imaging current (nA)</b>: .25<b>Scanning speed (MHz)</b>: .1<b>Dataset URL</b>: s3://janelia-cosem-datasets/jrc_hela-2/jrc_hela-2.zarr/recon-1/em/<b>Visualization Website</b>: https://openorganelle.janelia.org/datasets/jrc_hela-2<b>Publication</b>: Xu et al, 2021, Heinrich et al., 2021