Stable LLC-PK1 cell line (ATCC:CL-101) was generated and provided by Michael W. Davidson [1,2]. Using a Zeiss Celldiscoverer 7 microscope with a 100X/1.47 NA oil-immersion objective (Plan-Apochromat, Zeiss) and a sCMOS sensor (Hamamatsu, ORCA-Fusion, C15440-20UP), cultured LLC-PK1 cells expressing mEmerald-EB3 were imaged with a 43 nm pixel size. Exposure time was 100 ms, with each frame being captured every two seconds. During acquisition, temperature and CO2 control was set to 37°C and 5%, respectively. mEmerald-EB3 was excited using a 488 nm laser at 1% power, with a FITC filter for fluorescence collection. Acquisition software ZEN 3.2 (blue edition) was used for the imaging protocol. 1. Rizzo, M. A., Davidson, M. W., & Piston, D. W. (2009). Fluorescent protein tracking and detection: fluorescent protein structure and color variants. Cold Spring Harbor Protocols, 2009(12), pdb-top63. 2. Huang, F., Hartwich, T., Rivera-Molina, F. et al. Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms. Nat Methods 10, 653–658 (2013).