摘要The plasma membrane is a sophisticated,organized,and highly heterogeneous structure that compartmentalizes cellular processes.To decipher the biological processes involving membrane proteins,it is necessary to analyze their spatiotemporal dynamics.However,it is difficult to directly assess the dynamics and interactions of biomolecules in living cells using traditional biochemical methods.Singleparticle tracking (SPT) methods for imaging and tracking single particles conjugated with fluorescent probes offer an ideal approach to acquire valuable and complementary information about dynamic intracellular processes.SPT can be used to quantitatively monitor the diverse motions of individual particles in living cells.SPT also provides super-spatiotemporal resolution that allows early-stage or rapid response information to be obtained for a better understanding of molecular basis of associated signal transduction processes.More importantly,SPT can be used to detect the motion paths of individual biomolecules in vivo and in situ,thus unveiling the dynamic behavior of the biomolecules that support developmental processes in living cells.In this review,we give an overview of SPT methods,from image acquisition to the detection of single particles,as well as tracking and data analysis.We also discuss recent applications of SPT methods in the field of plant biology to reveal the complex biological functions of membrane proteins.