One of the overarching goals of Lakadamyali lab is to determine how molecular motors coordinate to transport vesicles and organelles in the complex cellular environment. To achieve this goal, we developed an “all-optical correlative imaging method” that combines single particle tracking in living cells with super-resolution microscopy of the same cell after fixation. Super-resolution microscopy can reveal the details of cellular architecture with exquisite spatial resolution; however, the current methods cannot capture fast dynamic processes (millisecond time scale) due to limited temporal resolution. With the correlative approach, we overcame this limitation and for the first time related cargo transport dynamics to the organization of the microtubule cytoskeleton in the cellular context. By mapping cargo trajectories to individual microtubules, we studied how the local cytoskeletal architecture regulates vesicle trafficking. We found that the cytoskeleton acts as a selective filter to vesicles that are comparable in size to or larger than the mesh size of the microtubule network leading to their pausing. We further showed that lysosomal and autophagosomal compartments are selectively enriched on specific microtubule tracks defined by post-translational modifications in order to enhance their fusion and regulate autophagy. Overall, we have developed powerful methods that reveal how the 3D local cytoskeletal architecture and the microtubule post-translational modifications combine to regulate, opening a new window into studying vesicle-cytoskeleton interactions in the physiological as well as disease contexts.