Zooming into Disease at the Nanoscale: High-Speed Atomic Force Microscopy in Biomedical Discovery.

The dynamic properties of biomolecules are important to the biogenesis of essential building blocks, the maintenance of biological homeostasis, and the cellular responses to external stimuli critical for survival. Structural studies define biomolecular functions, and their dynamic behavior provides mechanistic insights into these processes. Real-time tracing of dynamic biomolecular structural changes and interactions is highly desirable. Current methodologies such as cryo-EM and X-ray crystallography could provide detailed structural information; however, the images are rather static. Insufficient temporal resolution precludes the observation of crucial transient intermediates. Furthermore, sample preparation and non-physiological imaging environments can compromise the native state of biomolecules. On the other hand, other tools, such as FRET and NMR, could detect dynamic changes of targets at high temporal resolution but lack real-time observation. Atomic force microscopy (AFM) enables nanoimaging and biophysical characterization of biomolecules but is limited by slow scanning speeds and strong tapping forces. Later, high-speed AFM (HS-AFM) with high spatiotemporal resolution and gentle tapping forces, emerged as an ideal nanoimaging approach for studying the functions of delicate biological samples. Building on these developments, we and others have contributed to expand HS-AFM toward biomedical applications, including the direct visualization of disease-relevant organelles and nanostructures under near-physiological conditions. In this review, we examine HS-AFM applications in biomedical science, emphasizing real-time nanoimaging of structural dynamics across biological systems relevant to infectious diseases, infertility, cancer, and neurodegeneration. We also critically discuss the technical limitations of HS-AFM and mitigation strategies.