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.
words):Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells. modalities (supplementary note 1, table S1), allowing specific molecular components to be visualized at nanoscale resolution in the context of the crowded intracellular environment.However, SR/EM correlation often involves tradeoffs in sample preparation between the retention of fluorescent labels, sufficiently dense heavy metal staining for high contrast EM, and faithful preservation of ultrastructure, particularly when chemical fixation is used (19)(20)(21)(22).Here we describe a pipeline ( fig. S1) for correlative cryo-SR/FIB-SEM imaging of whole cells designed to address these issues. Specifically, cryogenic, as opposed to room temperature, SR performed after high pressure freezing (HPF), allowed us to use a standard EM sample preparation protocol without compromise. We used cryogenic 3D structured illumination (SIM) and single molecule localization (SMLM) microscopy for SR protein specific contrast with 3D FIB-SEM for global contrast of subcellular ultrastructure. The SR modality highlights features not readily apparent from the EM data alone, such as exceptionally long or convoluted endosomes, and permits unique classification of vesicles of like morphology, such as lysosomes, peroxisomes, and mitochondrial-derived vesicles. Cell-wide 3D correlation also reveals unexpected localization patterns of proteins, including intranuclear vesicles positive for an ER marker, intricate web-like structures of adhesion proteins at cell-cell junctions, and heterogeneity in euchromatin or heterochromatin recruitment of transcriptionally-associated histone H3.3 and heterochromatin protein 1α (HP1α) in the nuclei of neural progenitor cells as they transition into differentiated neurons). More generally, whole cell cryo-SR/FIB-SEM can reveal compartmentalized proteins within known subcellular components, help discover new subcellular components, and classify unknown EM morphologies and their roles in cell biology. Cryogenic SR below 10K: motivations and photophysical characterization
Light upconversion is a very powerful tool in bioimaging as
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.