Cryo-Electron Tomography (Cryo-ET) is an effective imaging technique used in structural biology. It allows scientists to visualize the three-dimensional (3D) structures of biological components, such as cells, viruses, and molecules, in their native, frozen state at extremely high resolution. This method provides detailed insights into the organization and function of cellular structures and has advanced our understanding of life’s fundamental processes. By preserving samples in a near-native environment, Cryo-ET helps researchers observe biological structures without the distortions often introduced by traditional preparation methods.
How Cryo-ET Works
Cryo-ET begins with vitrification, a specialized sample preparation technique. This involves rapid freezing to preserve biological samples in a near-native, hydrated state. This swift cooling, typically below -160°C using liquid nitrogen or liquid ethane, prevents water molecules from forming damaging ice crystals. Instead, it creates a glassy, amorphous ice that maintains the sample’s original structure. This vitrified state is important because ice crystals can disrupt delicate biological structures, introducing artifacts.
For thicker biological samples, such as eukaryotic cells or tissues, focused ion beam (FIB) milling is often necessary. Most cells are too thick for electrons to penetrate sufficiently. FIB milling uses a beam of gallium ions to remove material, creating thin, electron-transparent sections, known as lamellae, typically 100 to 250 nanometers thick. This process allows researchers to target specific regions of interest within a cell, ensuring the electron beam can pass through for imaging while preserving the cellular context.
Once the sample is prepared, a transmission electron microscope (TEM) is used for data acquisition. The vitrified sample is placed on a goniometric stage inside the TEM and progressively tilted to different angles relative to the electron beam. At each tilt angle, a two-dimensional (2D) projection image of the sample is recorded by a camera, generating a “tilt series.” This process is performed under low electron dose conditions to minimize radiation damage to the delicate biological material.
After the tilt series is collected, computational algorithms are used for 3D reconstruction. These algorithms align the 2D images from the tilt series, correcting for any shifts or distortions. They then computationally merge them to create a detailed 3D volume, known as a tomogram. This reconstruction process fills in missing information to create a comprehensive 3D representation of the sample. Software packages like IMOD and AuTom are used for these tasks, transforming 2D images into a 3D map.
Unveiling Biological Structures
Cryo-ET allows scientists to visualize and study a wide array of biological samples and structures with great detail. This includes observing cellular structures directly within intact cells, known as in situ imaging. Researchers can reveal the spatial arrangement and interactions of organelles, molecular complexes, and cytoskeletal elements, providing insights into their organization. For example, it has been used to study the endoplasmic reticulum-associated degradation machinery, revealing its location and organization.
The technique is also applied to viruses and pathogens, allowing for a deeper understanding of their structures, assembly processes, and interactions with host cells. Cryo-ET can visualize pleomorphic viruses and capture dynamic conformational changes during infection cycles. For instance, it has revealed features of viral RNA replication compartments within infected Drosophila cells, enhancing insights into their structure and function.
Cryo-ET is used in resolving the 3D architecture of macromolecular complexes, such as large protein complexes and ribosomes. It can capture snapshots of transient states or dynamic processes within cells, offering a perspective on molecular machines in action.
The Impact of Cryo-ET
Cryo-ET has advanced scientific research by offering advantages in visualizing biological structures. By avoiding chemical fixation and staining, which can introduce artifacts and alter native structures, Cryo-ET provides a more accurate representation of biological components in their natural, hydrated state. This preservation allows for the observation of delicate molecular interactions and arrangements as they exist within living systems.
A strength of Cryo-ET is its ability to provide high-resolution structural information within the complex cellular context. Unlike methods that require purified components, Cryo-ET can reveal how molecules and organelles interact in situ, offering insights that cannot be gained from studying isolated parts. This contextual information is valuable for understanding the spatial relationships and functional coordination of cellular machinery.
Insights gained from Cryo-ET also contribute to understanding the structural basis of various diseases, including neurodegenerative disorders and infectious diseases. By visualizing pathogens like viruses and bacteria within host cells, researchers can uncover mechanisms of infection and disease progression. This structural knowledge can guide the development of new therapeutic strategies by identifying specific molecular targets and aiding in the rational design of new medicines, for instance, by revealing the binding modes of potential drug compounds to protein targets.