Advances, challenges, and applications of cryo-electron tomography workflows for three-dimensional cellular imaging of infectious pathogens.

Cryo-electron tomography (cryo-ET) has become a powerful tool for visualising cellular structures at sub-nanometer resolution in their near-native state, offering unique insights into the molecular architecture of diverse biological systems, including infectious agents and their interactions with host cells. This paper reviews key methodologies and recent advancements in cryo-ET, with a particular focus on sample preparation of protozoan parasites and host cells. Topics covered include photopatterning for cell positioning on EM grids, vitrification techniques, whole-cell imaging, and cryo-FIB milling followed by cryo-ET. The manuscript also addresses how these approaches are providing valuable structural information on pathogens and pathogen-host interactions, which are critical for understanding mechanisms of pathogenesis and the development of therapeutic strategies. Additionally, we examine the principles and practical considerations of the multistep workflow, highlighting innovations such as integrated fluorescence microscopy (iFLM) within cryo-FIB SEM systems for improved target identification and lamella positioning. Challenges such as ion beam damage, sample thickness constraints, and the need for greater workflow automation are also discussed as areas for future improvement. As cryo-ET continues to evolve and deliver transformative insights into the molecular architecture of life, it inspires great hope for the development of future therapies against infectious diseases. LAY DESCRIPTION: Cryo-electron tomography (cryo-ET) is a special type of microscopy that allows researchers to look at the inside of cells in 3D, almost as if a hologram of the cell in its natural state was generated. This technique reveals molecular structures inside cells, allowing scientists to better understand how molecules and cellular components work together. To obtain such detailed images, biological samples need to be thin and frozen very quickly so that they remain undamaged and close to their natural state. One recent breakthrough involves using a tool called cryo-focused ion beam scanning electron microscopy (cryo-FIB SEM), which allows a thin slice of a frozen sample to be collected and then analysed using cryo-ET. In addition, photopatterning of support surfaces are being used to place cells in a strategic position for cryo-FIB SEM, and improved plunge freezing and high-pressure freezing methods have been developed to better preserve samples. Together, these techniques make it easier to reproducibly prepare high-quality samples for cryo-ET. These innovations allow capturing clearer and detailed images of cells, tissues, and even entire small organisms. Cryo-ET has led to important discoveries in biology, such as how proteins and other molecules interact within cells at the sub-nanometre scale. This technique holds great promise for revealing how life works at a molecular level, understanding diseases, and discovering new drugs.