Introduction to CryoET

"You can observe a lot by watching..." When it comes to biological machinery you can't see, CryoET promises to let researchers view the whole picture.

What is CryoET?

Cryo-electron tomography (CryoET) is a technique that provides researchers with a 3D view of cells, subcellular components (e.g., organelles), viruses, and proteins. Detailed 3D image reconstructions generated through CryoET enable researchers to answer questions that are critical for understanding cellular function and response, such as:

  • How are subcellular components and proteins spatially organized within a cell?

  • How do subcellular components and proteins interact with each other?

  • How do cells change in response to stress, injury, or disease?

  • How do infectious agents interact with host cells?

Cryo-electron tomogram of a sectioned spore from the intracellular parasite Encephalitozoon intestinalis. The video shows tomographic slices along the depth dimension of the volume (left) and voxel segmentations of various parts of the infection machinery (right). Encephalitozoon intestinalis is an opportunistic human pathogen that uses a harpoon-like apparatus called the polar tube to invade host cells. CryoET helped investigate the infection mechanism through the polar tube. When activated, the specialized polaroplast organelle swells and causes the polar tube to be expelled and catapulted through the host cell membrane, allowing injection of infectious material. This run is available on the CryoET data portal as part of dataset 10437 and was generously contributed by Usmani et. al as supporting material for their E. intestinalis study.

What makes CryoET special?

Thanks to CryoET, researchers can now bridge the gap between techniques that enable them to study cells at the micron scale (e.g., light microscopy) and molecular structures at the atomic-level (e.g., X-ray crystallography). Although there are other techniques that generate 3D models of subcellular components, viruses, and proteins, these techniques require isolation of single particles. CryoET is the only technique that enables researchers to study biological samples in their native environment at near atomic resolution while providing context about their location, conformation, and interactions within a cell or medium. It is a powerful technique that can provide structural information at a range of resolutions, from whole cells to molecules, including the atomic structure of particles with resolutions near 3 angstrom (Å; 1 Å = 0.1 nm).

Schematic of resolution ranges achieved by different microscopy and structural determination methods.
Schematic of resolution ranges achieved by different microscopy and structural determination methods. Icons highlight cells and biological components that can be captured at various resolution ranges. The scale bar depicts millimeter (mm) to angstrom (Å) level resolutions.

What is the technology behind CryoET?

The main technologies that make CryoET possible are highlighted in its name, including: cryogenic techniques, electron microscopy and tomography.

Cryo

Cryo refers to cryogenic freezing techniques that are used to fix samples without the use of chemical fixatives or stains. Cryo-fixation happens so quickly that biological material and processes are frozen in their hydrated, near native state before ice crystals start to form. This flash-freezing process that preserves the natural structure of the sample in a glass-like state is known as vitrification because it embeds the sample in amorphous (vitreous) ice. One of the first steps during CryoET sample preparation is to “vitrify” samples. Cryo-fixation also protects the sample when exposed to the high-vacuum environment of the electron microscope.

Schematic and electron microscopy images showing how vitrification preserves specimens in their native state and fine structural details.
Schematic showcasing how vitrification keeps cells in their hydrated, near-native state (top panel) and electron microscope (EM) images of the Golgi apparatus (bottom panel) from a chemically fixed cell (A) versus a CryoET reconstruction (B). Note that crystalline ice formation damages cell membranes and excludes solutes (e.g., salts and sugars) from the ice lattice, increasing solute concentrations to lethal levels and causing dehydration. When cells are vitrified, cell membranes and solutes remain in their original position as water transitions into a glass-like state that prevents molecular movement. When looking at the EM images, note the molecular cross bridges preserved in the cryoET reconstruction (yellow box) that can’t be observed in the chemically preserved sample. Chemically preserved samples lose fine biological details due to the staining process. The EM images were originally published in a review by Hylton and Swulius 2021 and portrayed in CryoET 101.

Electron

Electron specifies that CryoET is an electron microscopy technique where an electron beam interacts with the sample to project an image. Electron microscopy is used to view and gain structural information about subcellular and viral components. Electron wavelengths are small enough to interact with these components and produce images based on those interactions. CryoET falls under the transmission electron microscopy category, where electrons pass through the sample and illuminate film or a digital camera. High electron density components cast stronger shadows than lighter density ones, thus producing a 2D projection of the material in the sample. Click here for a video explaining how electron microscopes work.

Schematic of a transmission electron microscope.
Transmission electron microscope schematic highlighting components of the illumination and imaging systems and the electron detection chamber.

Tomography

Tomography refers to an imaging technique that provides 3D information of an object by capturing projection images from multiple angles. CryoET collects 2D images representing rotational views or tilted projections from a sample. The collected 2D images, known as a tilt series, are then transformed into volume providing spatial information. Reconstructed 3D images are known as tomograms.

Schematic of the CryoET imaging workflow.
Schematic of the CryoET imaging workflow. (A) First, a vitrified specimen, depicted here as a cell (gray oval) embedded in an ice slab is tilted at a range of angles while an electron beam passes through the sample producing projection images. The cell includes three distinct molecular components (red, blue, and green objects) (B) Projected images from rotational views around a common axis produce a tilt series. (C) Finally, the tilt series is computationally aligned and used to reconstruct a 3D map of the imaged specimen through back-projection. Image adapted from Galaz-Montoya and Ludtke 2017.

Computational efforts are continuously optimizing tomogram reconstruction (e.g., automation of image pre-processing steps, such as image alignments, and improving signal-to-noise ratios). High quality tomograms can then be used to computationally improve the resolution of smaller, repetitive particles within tomograms to reconstruct their structure. This single particle reconstruction from tomograms is known as subtomogram averaging. Through subtomogram averaging, CryoET data can lead to molecular structures with resolutions near 3 Å.

Schematic of the subtomogram averaging workflow.
Subtomogram averaging workflow schematic. During subtomogram selection, the volumes of repetitive particles representing the same cellular component are selected from reconstructed tomograms. In this example, repetitive particles are represented by the blue structures and red arrows indicate their orientations. Selected subtomograms are then aligned in a way that 3D structures can be overlaid for averaging. Averaged subtomograms result in high resolution 3D structures. Image adapted from Jonker 2020.