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oduction Cryogenic transmission electron microscopy (cryo-TEM) has had a major impact in solving structural biology questions as well as in aiding the investigation of other nano- and micro-sized biological samples, without the need for staining or chemical fixation. Rapid advances in cryo-TEM methodology have led to a revolution in three-dimensional (3D) structure determination of biological macromolecules at near-atomic resolution.1–4 Thousands of two-dimensional (2D) particle images selected from cryo-electron micrographs are averaged to produce the 3D structure using a method called single-particle reconstruction. This approach assumes that the sample particles have a relatively uniform structure, as is often the case in structural biology. Another powerful technique, cryo-electron tomography (cryoET), offers an alternative approach for generating 3D structures of samples with a significant degree of heterogeneity.5–7 The cryo-ET approach involves the collection of a tilt-series, or a set of 2D cryo-TEM images, of a particular region of the specimen viewed from multiple angles (e.g., ±60°). The images are then reconstructed to give a 3D representation of the sample, called a tomogram, which represents the density within the particle.

Cryo-TEM techniques have recently caught the attention of soft matter experimentalists. In order to characterize soft matter in its native state, investigations have thus far typically relied on ensemble-scattering techniques (e.g., dynamic light scattering or small-angle x-ray scattering). To obtain a realspace image using conventional TEM, soft matter requires some modification, such as negative staining using uranyl acetate, to increase the contrast of the carbon-based sample against the substrate. Not only does this introduce structural uncertainty, the sample still needs to be dried onto a substrate to ensure stability in a high-vacuum environment, a wellknown source of artifact inclusion. Liquid-cell TEM is a powerful technique that allows for capturing the dynamics of a particle system in solution. However, its use has been limited for soft matter research due to reduced resolution caused by the increased electron path length through the liquid, coupled with typical low contrast. Furthermore, the inherent particle dynamics in liquid cell TEM make 3D reconstruction and the imaging of interfaces difficult. Cryo-TEM, in contrast, “fixes” the sample in place, which eliminates the possibility of probing dynamics. This static

John Watt, Center for Integrated Nanotechnologies, Los Alamos National Laboratory, USA; [email protected] Dale L. Huber, Center for Integrated Nanotechnologies, Sandia National Laboratories, USA; [email protected] Phoebe L. Stewart, Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, USA; [email protected] doi:10.1557/mrs.2019.285

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