Cryogenic electron microscopy has emerged as a powerful technique for characterizing biological nanomaterials, enabling high-resolution structural analysis while preserving native conformations. The method involves rapid vitrification of samples to trap biomolecular structures in a near-native hydrated state, followed by imaging at cryogenic temperatures to minimize radiation damage. This approach provides unique advantages for studying protein assemblies, lipid-based nanoparticles, and DNA nanostructures without the need for crystallization or chemical fixation.

Vitrification represents the critical first step in cryo-EM sample preparation, where aqueous suspensions of biological nanomaterials are flash-frozen to form amorphous ice. The process typically employs ethane slush cooled by liquid nitrogen to achieve cooling rates exceeding 100,000 Kelvin per second, preventing ice crystal formation that could damage delicate nanostructures. Sample application methods have evolved to address challenges in preparing thin, homogeneous ice layers, with plunge-freezing devices now incorporating advanced features such as humidity control and automated blotting. For lipid nanoparticles and membrane protein complexes, the addition of surfactants or glycerol can improve vitrification quality, though optimization remains empirical for each nanomaterial system.

Low-dose imaging protocols form the foundation of successful cryo-EM data collection, balancing the need for sufficient signal with the imperative to minimize beam-induced damage. Modern instruments implement dose fractionation across multiple frames, typically limiting total exposure to 20-50 electrons per square angstrom. This approach preserves high-resolution information while distributing damage across multiple readouts. Automated acquisition systems now incorporate real-time particle detection and focusing routines, significantly improving throughput for heterogeneous biological nanomaterials. For radiation-sensitive specimens like DNA origami structures, dose rates below 5 electrons per pixel per second have shown improved preservation of fine structural details.

Three-dimensional reconstruction methods have undergone transformative improvements, driven by advances in detector technology and computational algorithms. Direct electron detectors with high quantum efficiency enable motion correction at the single-electron level, recovering high-resolution information previously lost to beam-induced specimen movement. Processing pipelines for single-particle analysis now routinely achieve sub-3Å resolution for well-behaved protein complexes, with specialized approaches reaching 1.5-2Å for highly symmetric viral particles. For flexible or heterogeneous nanomaterials like lipid nanoparticles, classification algorithms can separate structural variants and reconstruct them individually, providing insights into dynamic conformational states.

Protein-based nanostructures present both opportunities and challenges for cryo-EM characterization. Well-ordered assemblies such as viral capsids or engineered protein cages often yield high-resolution maps suitable for atomic model building. More flexible systems, including amyloid fibrils or intrinsically disordered protein complexes, require specialized processing approaches to extract structural information from heterogeneous datasets. Recent developments in Volta phase plate imaging have improved contrast for small protein complexes below 100 kDa, expanding the size range accessible to cryo-EM analysis. The technique has proven particularly valuable for characterizing post-translational modifications and bound ligands that might be disrupted by crystallization.

Lipid nanoparticles represent another important class of biological nanomaterials where cryo-EM provides unique structural insights. The technique can resolve lamellar, inverted hexagonal, and cubic phases within lipid assemblies, as well as characterize the encapsulation and distribution of therapeutic payloads. Challenges include beam sensitivity of unsaturated lipids and the low contrast between aqueous compartments and lipid bilayers. Tilt-series cryo-electron tomography has emerged as a powerful complement to single-particle approaches for these systems, providing three-dimensional context for irregular or polymorphic structures. Recent work has demonstrated the ability to resolve individual lipid headgroups and track the distribution of cholesterol molecules within nanoparticle formulations.

DNA origami and other nucleic acid nanostructures benefit from cryo-EM's ability to visualize non-periodic arrangements without averaging artifacts. The high negative charge density of DNA provides sufficient contrast for reliable particle picking, though the relatively uniform mass distribution along helices can complicate orientation determination. Cryo-EM has revealed detailed structural features of DNA junctions, crossover points, and bound proteins at resolutions sufficient to identify individual base pairs in some cases. For larger DNA origami structures, the technique can validate designed geometries and identify folding errors or mechanical stresses within the nanostructures.

Resolution improvements in cryo-EM have been dramatic, with several technological innovations contributing to this progress. Direct electron detectors eliminated the resolution limit imposed by scintillator-based systems, while improved stage stability reduced mechanical vibrations during imaging. Computational advances in contrast transfer function correction and Bayesian particle classification have further pushed resolution boundaries. Current limitations include intrinsic sample movement during irradiation and variability in ice thickness across the grid. For biological nanomaterials, heterogeneity in composition or conformation often represents the ultimate resolution barrier rather than instrumental factors.

Sample preparation remains the most persistent challenge in cryo-EM of biological nanomaterials. Issues such as particle adsorption to air-water interfaces, preferential orientation, and ice contamination continue to require empirical optimization for each new system. Recent developments in graphene support films and affinity grids show promise for addressing some of these challenges, particularly for small or low-contrast particles. Automated vitrification systems with environmental controls help improve reproducibility, though the art of grid preparation still often outweighs the science in determining experimental success.

The future of cryo-EM for biological nanomaterials lies in integrating these techniques with complementary approaches and pushing toward high-throughput analysis. Correlative light and electron microscopy allows targeted imaging of rare or transient nanostructures, while time-resolved cryo-EM methods begin to capture dynamic processes. As the field matures, standardization of sample preparation protocols and data processing pipelines will be essential for translating cryo-EM from a specialized technique to a routine characterization tool for biological nanomaterials across research and industrial applications. The continued development of automated workflows and improved detectors promises to further expand the scope and accessibility of this transformative technology.