High-speed atomic force microscopy (HS-AFM) enables real-time visualization of biological processes with nanometer-level resolution. This review highlights how HS-AFM has been instrumental in uncovering the dynamic interplay between nuclear pore complexes (NPCs)-which regulate nucleocytoplasmic transport-and genome guardians, including DNA repair proteins and chromatin regulators. Structurally, the NPCs resemble a multi-layered spider cobweb, serving as crucial molecular gatekeepers for maintaining cellular homeostasis, while genome guardians safeguard genomic integrity through DNA repair and chromatin organization. Through HS-AFM, the researchers have gained unprecedented insights into NPC dynamics, revealing their adaptability during nuclear transport, chromatin reorganization, and viral infection. It has also elucidated how genome guardians interact with NPCs, influencing chromatin organization at the nuclear periphery and regulating nucleocytoplasmic trafficking. These discoveries underscore the critical role of NPC-genome interactions in genome stability, gene expression, and nuclear transport, with broad implications for diseases such as cancer, viral infections, and neurodegenerative disorders. In conclusion, HS-AFM has transformed our ability to study the nuclear landscape at the nanoscale, bridging the gap between structural biology and functional genomics. By capturing the real-time molecular dynamics of NPCs and chromatin, HS-AFM provides an essential tool for unraveling the mechanisms that govern nuclear transport and genome regulation. Future advancements in HS-AFM technology, including higher temporal resolution, correlative imaging, and AI-driven analysis, will further expand its potential in biomedical research, paving the way for novel diagnostic and therapeutic strategies.