Cryo-electron tomography (CryoET) is an imaging technique that has transformed our ability to visualize biological structures in detail. This method allows scientists to observe molecules, organelles, and even entire cells in their natural, three-dimensional state. By providing highly detailed views of the machinery of life, CryoET offers insights into how these complex systems function. This capability is expanding our understanding of biological processes at a molecular level.
The Core Principles of CryoET
CryoET relies on vitrification to preserve the sample’s natural structure. Biological samples are rapidly frozen to extremely low temperatures, typically below -160°C, using liquid nitrogen or liquid ethane. This rapid cooling turns the water within the sample into a glass-like solid, known as vitreous ice, preventing damaging ice crystals that would disrupt delicate cellular components. This method avoids artifacts introduced by chemical fixation or dehydration.
Once vitrified, the sample is transferred to an electron microscope. Unlike light microscopes, electron microscopes use a beam of electrons to image samples. Electrons have a much shorter wavelength than light, allowing for significantly higher resolution and the visualization of much smaller structures, down to the nanoscale. As electrons pass through the thin, frozen sample, they interact with the material, producing a two-dimensional projection image.
To achieve a three-dimensional view, CryoET uses electron tomography. This involves collecting multiple 2D images as the sample is tilted at various angles, typically from about -60 to +60 degrees. This series of tilted images, known as a tilt series, captures different perspectives of the same structure. Computational algorithms then combine these 2D images to reconstruct a detailed 3D representation, similar to how a medical CT scan builds a 3D image. For effective imaging, samples generally need to be very thin. Thicker specimens, such as whole cells, may require thinning using techniques like focused ion beam (FIB) milling to create a thin lamella.
What CryoET Reveals About Cells
CryoET provides insights into the architecture and function of biological structures directly within their cellular context. It allows researchers to visualize macromolecular assemblies, cellular organelles, and even entire cells in a near-native state. This “in situ” visualization reveals how these components interact and are organized within the crowded cellular environment, a level of detail previously unattainable.
The technique has been instrumental in studying various cellular processes and structures. For instance, it has been used to analyze the structure of the nuclear pore complex, a large protein assembly that regulates transport across the nuclear envelope. CryoET has also illuminated the organization and dynamics of the cytoskeleton, observing its morphology during interactions with viruses and parasites.
Beyond static structures, CryoET can capture different conformational states of proteins, providing clues about their dynamic behavior and functional mechanisms. This allows scientists to observe molecular machines in action, such as how viruses interact with host cells. Recent discoveries include the identification of previously unknown cellular structures, like the “hemifusome,” a membrane-bound organelle involved in cellular sorting and recycling.
The Importance of CryoET
CryoET represents a significant advancement in biological imaging because it allows for the visualization of biological samples in their near-native state. This capability avoids distortions and artifacts that can arise from traditional sample preparation methods, such as chemical fixation and staining, leading to a more accurate representation of cellular reality.
The technique’s capacity to deliver high-resolution 3D information directly from cells enhances our understanding of fundamental biological processes. It reveals how individual proteins interact and organize within the complex cellular environment. This detailed structural information helps decipher the mechanisms of various biological functions and disease processes.
The insights provided by CryoET hold promise for accelerating drug discovery and therapeutic development. By visualizing the binding modes of small molecules to their target proteins, the technique can inform the design of new compounds. This direct observation of molecular targets within their biological context aids in developing more effective treatments.