Advances in super-resolution imaging technology bring new perspectives to cytoskeleton mechanism research

The cytoskeleton constitutes the fundamental structural framework governing cellular morphology and the regulation of intracellular cellular processes. Its nanoscale architecture and dynamics are essential for understanding the mechanisms of fundamental biological activities. However, conventional imaging techniques are constrained by Abbe diffraction limit, precluding the visualization of cytoskeleton at nanoscale. While electron microscopy (EM) offers superior resolution, it is intrinsically constrained by a lack of live-cell compatibility and restricted three-dimensional (3D) resolving power. These technical bottlenecks have long hindered the in-depth dissection of cytoskeleton regulatory mechanisms. Super-resolution microscopy (SRM) has emerged as transformative paradigm, circumventing the diffraction barrier to enable high-resolution imaging of ultrastructural components and real-time tracking within living systems. This review provides a comprehensive synthesis of the development of mainstream SRM modalities-especially Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), and Single-Molecule Localization Microscopy (SMLM)-and their applications in investigating microfilaments, microtubules, and intermediate filaments. We delineate key biological insights facilitated by SRM, including the discovery of the membrane-associated periodic skeleton (MPS) in axons, the vimentin-mediated stabilization of microtubule dynamics, and the unimpeded walking mechanism of kinesin-1 resolved via Minimum Fluorescence Flux Microscopy (MINFLUX) nanoscopy. Furthermore, we provide a comparative analysis of the performance metrics and application niches of various SRM techniques. By summarizing SRM-driven advancements and addressing current technical issues, this work serves as a critical reference for researchers navigating SRM selection to conduct innovative cytoskeletal research.