Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells
David P. Hoffman, Gleb Shtengel, C. Shan Xu, Kirby R. Campbell, Melanie Freeman, Lei Wang, Daniel E. Milkie, H. Amalia Pasolli, Nirmala Iyer, John Bogovic, Daniel R. Stabley, Abbas Shirinifard, Song Pang, D. R. Peale, Kathy Schaefer, Wim Pomp, Chi‐Lun Chang, Jennifer Lippincott‐Schwartz, Tomas Kirchhausen, David J. Solecki, Eric Betzig, Harald F. Hess
Abstract
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.