Cryo-Electron Microscopy and Cryo-Electron Tomography

High resolution images of proteins, assemblies, and organization in cells and tissues

Neutron imaging of water uptake under stress.

Cryo-electron microscopy (cryo-EM) helps reveal the elusive mechanism of transmembrane proton transport. Cryo-EM determined the structure of a yeast vacuole V_o proton channel pump complex at 2.7 Å resolution (15 polypeptides shown in different colors), specific protein-lipid interactions, and the role of water in the dynamic process. [PDB ID: 6MOR. Roh, S. H., et al. 2020. "Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of V_o complex," Science Advances 6(41), eabb9605. DOI: 10.1126/sciadv.abb9605.]

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) techniques use electrons to provide images of biological materials frozen in their native state. Materials range from proteins and nucleic acids to very large biological assemblies and complexes, and image resolutions range from nanometer to atomic scales.

Collectively a suite of sample preparation techniques, 200-300 kV electron microscopes, and data analysis tools is available at DOE user facilities to address a range of imaging needs for BER-mission-relevant biological research. Cryo-EM uniquely captures dynamic ensembles of macromolecular structures as they occur in solution. Image analysis can then separate these ensembles into high-resolution snapshots that capture their compositional and conformational variance and their dynamics.

Cryo-ET is an emerging technique that can resolve subcellular structures inside cells and tissues. It can achieve nanometer-scale resolution for the entire sample and atomic-scale resolution for abundant molecular components in situ when post-tomographic data processing is added. Cryo-fluorescence light microscopy (cryo-FLM) and subsequent cryo-ET of frozen, hydrated cells can be used to label specific proteins and study cellular and molecular functions and dynamics in the 3D context of cells and tissues at a higher resolution than any other imaging techniques. Cryo-ET can also be preceded by cryogenic focused ion beam scanning electron microscopy (cryo-FIB-SEM), to produce lamellae from vitrified cells that are thin enough for cryo-ET imaging.

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. A pipeline integrating three imaging modalities revealed the distribution of subcellular structures in yeast cells: cryo-fluorescence confocal microscopy, volume cryogenic focused ion beam scanning electron microscopy (volume cryo-FIB-SEM), and transmission cryo-ET. Cell membrane and wall (yellow) enclose the nucleus (orange), mitochondria (purple), vacuoles (red), and other subcellular structures. [Reprinted with permission from Elsevier from Wu, G-H., et al. 2020. "Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells," Structure 28(11) 1232–37. DOI: 10.1016/j.str.2020.07.017. Copyright 2020.]

Key Features of Cryo-EM/ET Imaging

Accommodates sample sizes ranging from tens of kilodaltons of biological molecules to viruses, parasites, bacteria, and eukaryotic cells and tissues.
All biological samples are embedded in vitrified ice without any chemical fixative.
Structure resolution ranges from nanometers to ångströms.
Cryo-EM structures of biochemically purified biomolecules and molecular machines can resolve individual atoms, water, and ions.
Cryo-ET structures of cells can resolve organelles and subcellular components at nanometer resolution, and macromolecular structures at near-atomic resolution.
Cryo-FLM can detect a fluorescent protein fused with a target of interest and correlate it with its cellular environment using Cryo-ET.
Microcrystal electron diffraction (micro-ED), a cryo-EM technique, can determine the atomic structure of small molecules or proteins from nanoscale 3D crystals.

BER Researchers Use Cryo-EM/ET to Study:

Viruses, bacteria, fungi, free-living algae, plant tissues, and cells under normal and environmentally stressed conditions
Root hairs and mycorrhizae, which play significant roles in plant nutrient exchange
Model soil systems with microbe-fungi interactions under different environmental conditions
Stream sediments and colloids (including iron colloids, the basis of nutrient transport and bog iron formation)
Natural products and metabolite structures

See more examples in Science Highlights

Sample Considerations

Accommodates biochemically purified biomolecules with molecular weights larger than ~28 kDa under different chemical conditions.
Cell environments in standard growth or culture conditions are preserved until the moment of plunge-freezing, which occurs on a millisecond timescale.
Ideal samples are less than 0.4 microns thick, either naturally or thinned by cryo-FIB or ultramicrotomy.
Samples thinner than 10 microns can be plunge-frozen, then thinned by cryo-FIB.
Tissue samples up to 200 microns thick can be high-pressure frozen, then cryo-sectioned by ultramicrotomy and thinned by cryo-FIB.
The standard sample support is a 3mm diameter electron microscope grid. Other types of sample supports may be accommodated.

Instruments at DOE User Facilities

Each instrument has unique characteristics. To determine the user facility and instrument best suited to your science questions, see additional information and contacts at the links below.

Laboratory for Biomolecular Structure (LBMS) — Brookhaven National Laboratory. Provides peer-reviewed research access, support, and training for the use of both high-end and screening cryo-EM.
Environmental Molecular Sciences Laboratory (EMSL) — Pacific Northwest National Laboratory. Multiple DOE-funded microscopes are available. EMSL periodically issues calls for user proposals from researchers interested in using cryo-EM and other capabilities (e.g., native mass spectrometry, fluorescence). Proposals for the use of only cryo-EM/ET or micro-ED may undergo an accelerated review and access process.
Stanford-SLAC Cryo-EM Center — SLAC National Accelerator Laboratory. Multiple instruments for cryo-EM, cryo-ET, cryo-FIB-SEM, and cryo-FLM are available. Some of these facilities are accessible after a merit-based proposal evaluation by an expert panel. Training is open to global scientific investigators.