BackgroundThe plasma membrane (PM) proteins function in a highly dynamic state, including protein trafficking and protein homeostasis, to regulate various biological processes. The dwell time and colocalization of PM proteins are considered to be two important dynamic features determining endocytosis and protein interactions, respectively. Dwell-time and colocalization detected using traditional fluorescence microscope techniques are often misestimated due to bulk measurement. In particular, analyzing these two features of PM proteins at the single-molecule level with spatiotemporal continuity in plant cells remains greatly challenging.ResultsWe developed a single molecular (SM) kymograph method, which is based on variable angle-total internal reflection fluorescence microscopy (VA-TIRFM) observation and single-particle (co-)tracking (SPT) analysis, to accurately analyze the dwell time and colocalization of PM proteins in a spatial and temporal manner. Furthermore, we selected two PM proteins with distinct dynamic behaviors, including AtRGS1 (Arabidopsis regulator of G protein signaling 1) and AtREM1.3 (Arabidopsis remorin 1.3), to analyze their dwell time and colocalization upon jasmonate (JA) treatment by SM kymography. First, we established new 3D (2D+t) images to view all trajectories of the interest protein by rotating these images, and then we chose the appropriate point without changing the trajectory for further analysis. Upon JA treatment, the path lines of AtRGS1-YFP appeared curved and short, while the horizontal lines of mCherry-AtREM1.3 demonstrated limited changes, indicating that JA might initiate the endocytosis of AtRGS1. Analysis of transgenic seedlings coexpressing AtRGS1-YFP/mCherry-AtREM1.3 revealed that JA induces a change in the trajectory of AtRGS1-YFP, which then merges into the kymography line of mCherry-AtREM1.3, implying that JA increases the colocalization degree between AtRGS1 and AtREM1.3 on the PM. These results illustrate that different types of PM proteins exhibit specific dynamic features in line with their corresponding functions.ConclusionsThe SM-kymograph method provides new insight into quantitively analyzing the dwell time and correlation degree of PM proteins at the single-molecule level in living plant cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13007-023-01047-5.