Tracking Small Extracellular Vesicles

The trajectories of sEVs were reconstructed from the original video stack with Imaging‐Pro‐Plus software (Media Cybernetics) by aligning the proximity and similarity in coordinate and intensity of each spots representing sEVs or corresponding structures in every frame. Only the trajectories within the focal plane were processed for quantitative analysis of single particle tracking. The MSD was calculated by the user‐written program with MATLAB software (MathWorks). The different motion modes were determined by fitting the dependence of MSD against time interval (Δt) with the formulas as below: MSD = 4DΔt + constant (free diffusion), MSD = 4DΔt^α + constant (restricted diffusion) and MSD = 4DΔt + (VΔt)² + constant (directed diffusion), where D and V represents the diffusion coefficient and the mean velocity respectively. The orthogonal slice view was obtained by UltraVIEW (PerkinElmer) to display the relative position of particles and cell in a 3D view. Kymograph analysis was performed with Image J. Line profile of colocalization or endocytosis were exhibited by using Imaging‐Pro Plus software.