Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells

Visualizing whole cells at many scales Cells need to compartmentalize thousands of distinct proteins, but the nanoscale spatial relationship of many proteins to overall intracellular ultrastructure remains poorly understood. Correlated light and electron microscopy approaches can help. Hoffman et al. combined cryogenic super-resolution fluorescence microscopy and focused ion beam–milling scanning electron microscopy to visualize protein-ultrastructure relationships in three dimensions across whole cells. The fusion of the two imaging modalities enabled identification and three-dimensional segmentation of morphologically complex structures within the crowded intracellular environment. The researchers observed unexpected relationships within a variety of cell types, including a web-like protein adhesion network between juxtaposed cerebellar granule neurons. Science, this issue p. eaaz5357 Cryogenic super-resolution fluorescence and electron microscopy reveals protein-ultrastructure relationships in whole cells. INTRODUCTION Our textbook understanding of the nanoscale organization of the cell and its relationship to the thousands of proteins that drive cellular metabolism comes largely from a synthesis of biochemistry, molecular biology, and electron microscopy, and is therefore speculative in its details. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) promises to elucidate these details by directly visualizing the nanoscale relationship of specific proteins in the context of the global cellular ultrastructure. However, to date such correlative imaging has involved compromises with respect to ultrastructure preservation and imaging sensitivity, resolution, and/or field of view. RATIONALE We developed a pipeline to (i) preserve fluorescently labeled, cultured mammalian cells in vitreous ice; (ii) image selected cells in their entirety below 10 K by multicolor three-dimensional structured illumination (3D SIM) and single-molecule localization microscopy (SMLM); (iii) image the same cells by 3D focused ion beam scanning EM (FIB-SEM) at 4- or 8-nm isotropic resolution; and (iv) register all image volumes to nanoscale precision. The pipeline ensures accurate ultrastructure preservation, permits independent optimization of SR and EM imaging modalities, and provides a comprehensive view of how specific subcellular components vary across the cellular volume. RESULTS Nearly every system we studied revealed unexpected results: intranuclear vesicles positive for a marker of the endoplasmic reticulum; peroxisomes of increasingly irregular morphology with increasing size; endolysosomal compartments of exceptionally diverse and convoluted morphology; a web-like adhesion network between cerebellar granule neurons; and classically EM-defined domains of heterochromatin and euchromatin each sub-characterized by the presence or absence of markers of transcriptional activity. Two-color cryo-SMLM enabled whole-cell image registration quantifiable down to ~40 nm accuracy. Cryo-SIM, even with its lower resolution, enabled unique discrimination between vesicles of like morphology and aided in segmenting complex 3D structures at FIB-SEM resolution within the crowded intracellular milieu. CONCLUSION Our pipeline serves as a powerful hypothesis generator to better understand the findings of biochemistry in the context of the spatially compartmentalized cell. Our approach also carefully preserves the native ultrastructure upon which such hypotheses are based, thus enabling cell-wide or cell-to-cell investigation of the natural variability in protein-ultrastructure relationships. Whole-cell correlative imaging. Cryogenic super-resolution fluorescence microscopy of high-pressure frozen cells coupled with focused ion beam scanning electron microscopy (FIB-SEM) enables multicolor three-dimensional nanoscale visualization of proteins in the context of global ultrastructure. Clockwise from upper left: Volume-rendered cell with correlated orthoslice (inset) of mitochondria and endoplasmic reticulum (ER) proteins; endolysosomal compartments of diverse morphology; heterochomatin subdomains defined by protein reporters of transcriptional activity; adhesion proteins correlated to membrane roughness at contacting cerebellar granule neurons; and a peroxisome (pink) juxtaposed to an ER sheet (red) and mitochondrion (cyan). 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.