Acoustofluidic cryo-EM enables in situ particle manipulation for uniform, high-quality cryo-EM specimens

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling imaging of macromolecular complexes at near-atomic resolution, but its success depends critically on specimen quality. Standard plunge freezing often produces nonuniform particle distributions, resulting in poor particle statistics and reduced imaging quality, particularly for vesicular structures and low-concentration, labile macromolecular complexes. Here, we introduce acoustofluidic cryo-EM (ACE), a noncontact specimen-preparation method that integrates acoustofluidic manipulation into a conventional plunge-freezing workflow. ACE enables localized nanoscale particle manipulation by mechanically coupling acoustic excitation into the cryo-EM grid, where forced vibration generates localized acoustic streaming through viscous boundary-layer effects. Guided by this physical mechanism, validation using liposomes and apoferritin demonstrates that ACE increases particle density by up to fourfold while preserving structural integrity and improving imaging quality. This strategy provides a broadly applicable approach for enhancing cryo-EM specimen quality, improving imaging efficiency, and facilitating high-resolution structural analysis.