From Sub-Cellular to Whole-Cell: Biological Imaging With Cryo-EM
Developments in cryo-electron microscopy are set to transform biological imaging by supporting more targeted analysis.
Life science research relies upon advanced imaging technologies for detailed insights into the structure and function of organs, tissues, cells, and sub-cellular molecules. High-resolution 3D images of biological samples provided by these technologies have the potential to unlock new discoveries and accelerate research across a range of applications, including disease diagnosis and monitoring, drug discovery and development, and environmental analysis.
Recent developments in cryo-electron microscopy (cryo-EM) are set to transform biological imaging by supporting more targeted analysis. With new cryo-EM systems, researchers can not only determine the structures of single molecules but can also image entire cells in their natural environments.
Cryo-EM at the forefront
In 2017, Jacques Dubochet, Joachim Frank, and Richard Henderson were awarded the Nobel Prize in Chemistry for their role in developing high-resolution cryo-EM for the structural determination of biomolecules.1 Prior to this, X-ray crystallography and nuclear magnetic resonance (NMR) were commonly used for biological imaging; however, both methods presented significant challenges. For example, X-ray crystallography samples must be crystallizable, limiting the types of samples that can be analyzed, and larger molecules can be difficult to distinguish using NMR.
The Nobel Prize award marked the emergence of cryo-EM as an effective and versatile alternative. In cryo-EM, biological samples are rapidly frozen to temperatures below -195 °C and then imaged from multiple angles using an electron microscope. The images are subsequently used to construct a 3D model of the sample at close to atomic resolution.
Several techniques have evolved from cryo-EM, including cryo-transmission electron microscopy (cryo-TEM), cryo-scanning electron microscopy (cryo-SEM), and cryo-focused ion beam-SEM (cryo-FIB-SEM). Researchers have benefited from the adoption of cryo-EM as it provides images of molecular structures in their native states, allowing them to acquire a more in-depth understanding of their samples. Unlike NMR, cryo-EM can easily handle larger molecules and, in a significant advancement over X-ray crystallography, molecules do not need to be crystallized before imaging.
As the technology develops, cryo-EM is fast becoming the go-to technique for biological imaging and, by the end of 2025, the number of samples analyzed using this method is set to have overtaken those analyzed by X-ray crystallography.2
Targeted analysis through collaboration
The Centre for Ultrastructural Imaging (CUI) at King’s College London, United Kingdom, is an EM facility supporting both internal and external collaborators across fields including medicine, chemistry, and biological sciences. Recently, the team collaborated with Linkam Scientific Instruments to develop a stage that can support cryo-FIB and cryo-TEM applications, while addressing the needs of researchers for more targeted analysis by offering improvements in the user interface and sample transfer capability.
A cryo-correlative light and electron microscopy (CLEM) stage has been optimized to help target regions of interest for cryo-FIB and cryo-TEM systems. The CUI is using the stage for cryo-FIB lamella preparation and has also been working on developing cryo-volume imaging for sectioning inside a cryo-FIB system, using the cryo-CLEM stage to help target cells. The stage has allowed the CUI to be more targeted in its analysis of biological materials, while preserving the sample’s native environment.
Cryo-TEM employs vitrification to solve near-atomic-resolution structures of a sample in close to its living state. The CUI’s cryo-TEM system, which utilizes the cryo-CLEM stage, has played an important role in the study of Plasmodium falciparum, a parasite that causes severe and often fatal malaria in humans. It provides high-resolution images of P. falciparum red blood cells during invasion, infection, and egress.
Alongside targeted analysis, cryo-FIB volume imaging, supported by the cryo-CLEM stage, opens up the possibility for observing larger structures. Vitrified samples undergo serial milling and imaging to produce a detailed 3D reconstruction of the cell’s internal organization, revealing organelles, membranes, and molecular details at nanoscale resolution. With cryo-FIB, researchers can study cells in their near-native state and bridge the gap between molecular details and the entire cellular context.
The future of cryo-EM
Having already supported significant advancements in life science research, cryo-EM has a promising future ahead. Research institutes are exploring the potential of cryo-electron tomography (cryo-ET) for imaging large volumes of samples.
Unlike conventional cryo-EM, molecules can be imaged inside a cell or a membrane, allowing researchers to view the sample much closer to its native state. Emerging technologies such as this, in addition to ongoing developments, are expediting biological imaging research timelines and lamella production.
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