Electron cryotomography is an emerging technique that allows the structures of unique biological objects such as individual macromolecules, viruses, and even small whole cells to be reconstructed in their near-native states in three dimensions (3-D) to an approximate 5-nm resolution. The required instrumentation, sample preparation and limitations, data collection, typical results, and future prospects are summarized briefly.

Introduction

The cell is a complicated factory with ordered and regulated assembly lines whose activities are performed by macromolecular assemblies that can rightly be called machines (1). An important goal is to understand the structures and functions of these machines and then eventually manipulate them. While X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are the most widely used tools of structural biology and can yield atomic models, they are limited to specimens that can be purified in high numbers, are conformationally homogeneous, and either crystallize or remain stable in solution in high concentrations for long periods of time. Likewise, electron cryomicroscopy-based single particle analysis techniques can generate medium-resolution (0.5–2 nm) reconstructions of large complexes, but again only if large numbers of structurally homogenous particles can be purified. Light microscopy can visualize unique objects, including living cells, but only to a resolution of approximately 400 nm. Electron cryotomography promises to fill the resolution gap in between, allowing the structures of unique macromolecular machines and supramolecular assemblies like viruses, organelles, or even intact small cells to be visualized to an approximate 5-nm resolution in a near-native state (2,3,4,5).

What Is ECT?

Electron cryotomography (ECT, or alternatively cryoelectron tomography) is the combination of three core technologies. Tomography refers to a generic imaging strategy wherein a higher-dimensional reconstruction of an object is calculated from a series of lower-dimensional projections. Most people are familiar with its medical application, the computerized axial tomography (CAT) scan, where two-dimensional (2-D) projections of patients are recorded with X-rays and then a three-dimensional (3-D) model is calculated and analyzed on a computer. In electron tomography, electrons are used to produce the projection images in a transmission electron microscope, with far higher resolutions (appropriate for individual macromolecules) but also far less specimen penetration. The cryo prefix refers to the fact that the sample is kept cold while it is being imaged, typically after being either plunge- or high-pressure frozen. Such directly frozen samples are preserved in a more native state than the typically chemical fixed, dehydrated, resin-embedded, sectioned, and stained samples used in traditional electron microscopy, but they are also more radiation sensitive and have poorer contrast.

What Is Needed to Do ECT?

Several expensive enhancements to standard electron microscopes are needed for ECT. First, in order to keep the (vitreously) frozen water in the sample from crystallizing, stages that keep the sample cooled to approximately −165°C or less are needed. The first generation of cryostages, which are still in frequent use today, have liquid nitrogen dewars attached to the end of the specimen rod. Because these require frequent refilling and suffer from drift and mechanical vibrations as the nitrogen boils away, more recently, large dewars have been mounted on the microscope and connected thermally to internal cartridges that hold the electron microscopy grids. Some of these specimen holders allow the grid to be rotated 90°C after one round of tilting to enable dual-axis tomography (6,7). The stage goniometer must be mechanically precise and eucentric so target regions do not move out of the beam when the sample is tilted to approximately ±70°C.

Second, because inelastically scattered electrons damage the sample and do not contribute usefully to images, samples should not be much thicker than the mean inelastic free path of an electron, which for a 300−keV electron in organic materials is approximately 0.35 µm. Thus higher-than-normal microscope voltages (200–400 kV) are typically used to balance higher specimen penetration against losses in electron detectability. Energy filters are also used to remove lower energy, inelastically scattered electrons from the beam, which would otherwise simply add noise to the image (8,9). Finally, charge-coupled device (CCD) cameras are necessary to allow real-time tracking and focusing during automatic data collection.

How Is ECT Performed?

To prepare a sample for ECT, a drop of purified protein, virus suspension, or cell culture is typically placed upon an electron microscopy grid which is then blotted and plunged into a cryogen (10). When the sample is appropriately thin and the plunging is sufficiently fast, liquid water in the sample is frozen into vitreous ice, preserving the sample in a near-native, frozen-hydrated state. Automated plunge freezers are now available for this crucial step to improve control and reproducibility (11,12), but manual blotting can still be necessary for fragile samples (13). Physiologically relevant buffers can be used (14,15). Cells can be grown directly upon the grid and then frozen (16). Plunge-frozen samples are preserved so well, in fact, that enzymes, viruses, and even cells have been seen to remain active, infective, or vital after thawing.