Double-Color MINFLUX Imaging of Microtubules and Vesicles

Microtubules and vesicles play key roles in intracellular transport and signal transduction. Accurately characterizing their positional relationship is crucial for understanding dynamic cellular processes. However, due to limited spatial resolution, conventional optical microscopy cannot clearly resolve their subcellular structures. MINFLUX super-resolution microscopy enables nanometer-scale localization precision in single-molecule imaging. This capability provides a useful tool for studying the positional relationship between microtubules and vesicles. In this study, MINFLUX was used to perform two-dimensional (2D) and three-dimensional (3D) imaging of microtubules and vesicles in BSC-1 cells, exploring their spatial relationships within the cytoskeletal framework. Our experimental results showed that MINFLUX imaging achieved nanometer-scale localization precision in both 2D and 3D. In 2D imaging, the system reached a localization precision of approximately 4 nm, while in 3D, the precision was around 6 nm. Furthermore, we used the detector channel ratio (DCR) derived from the dual-channel detection system to separate microtubule- and vesicle-associated localizations, enabling their differential assignment within the same MINFLUX measurement and revealing their relative spatial relationship within the cytoskeletal network.