Spatial Attributes of Actin Filaments Tethered by Myosin Fragments In Vitro Observed Using a Super‐Resolution Microscope

To evaluate whether nanoscale configurational changes in cytoskeletal filaments can be detected, we imaged AZDye532-labeled actin filaments bound to various myosin fragments in vitro using a home-built, low-cost direct stochastic optical reconstruction microscopy (dSTORM) system. The microscope, constructed by modifying a conventional fluorescence setup with a multimode 532-nm laser, achieved a spatial resolution of 25 nm. In the absence of ATP, actin filaments tethered to myosin subfragment-1 (S1) immobilized on a collodion-coated glass surface exhibited an average full width at half maximum (FWHM) of 38 nm. Replacing S1 with full-length myosin increased the FWHM to approximately 68 nm, indicating that the super-resolution images capture size-dependent configurational differences arising from thermal fluctuations of the tethering molecules. Desmin intermediate filaments labeled at their single cysteine residue with Alexa Fluor 532 maleimide were also imaged at super-resolution. Their transverse FWHM averaged 65 nm, and partially repeating axial intensity peaks with a spacing of approximately 45 nm were detected. These results suggest that periodic structural elements within intermediate filaments can be visualized under appropriate conditions. Overall, these findings demonstrate that an accessible dSTORM setup provides a practical platform for detecting nanoscale configurational changes in cytoskeletal filaments interacting with related proteins.