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Isotropic 3D electron microscopy reference data of COS-7 cell overexpressing ER and mitochondria markers for correlative light and electron microscopy studies (jrc_cos7-11)
Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
Sample: COS-7 cell overexpressing mEmerald-ER3 and Halo/JF525-TOMM20 (ATCC CRL-1651)
Protocol: High pressure freezing, freeze-substitution resin embedding with 2% OsO4 0.1% UA 3% H2O in acetone; resin embedding in Eponate 12.
Contributions: Sample provided by Melanie Freeman (HHMI/Janelia), prepared for imaging by Gleb Shtengel (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).
Dataset ID: jrc_cos7-11
Final voxel size (nm): 8.0 x 8.0 x 8.0 (X, Y, Z)
Dimensions (µm): 70 x 8 x 100 (X, Y, Z)
Acquisition date: 2018-04-27
Dataset URL: https://data.janelia.org/X2jWw
Visualization Website: https://openorganelle.janelia.org/datasets/jrc_cos7-11
Publication: Hoffman et al., 2020 (10.1126/science.aaz5357)