Fig. 3: Resolution of spatiotemporal clustering in live-cell molecular trajectory data.

Sx1a-mEos2 sptPALM data acquired at 50 Hz over 320 s. Clustering using NASTIC using r = 1.2, t = 20 s. a Raw acquisition data showing all molecular detections, with the region of interest (ROI) highlighted in yellow. b Spatiotemporal clustering of the selected trajectories within the ROI of (a), with a region highlighted with a dotted white box for enlargement in (c). c Enlargement of highlighted area in (b) showing individual trajectories and their centroids, with clusters highlighted and color-coded according to their time in the acquisition. The dotted box highlights a hotspot of repeated clustering. d 2D Kernel density estimation of the detections associated with the selected trajectories, with brighter blobs corresponding to higher density. e Instantaneous diffusion coefficient, with each trajectory colored according to the gradient of the first four time points in its mean square displacement (MSD). f 3D plot of the selected trajectories, rotated to show the temporal separation of the clusters highlighted in (c). g 1D plot of the selected trajectories where each vertical bar represents a single trajectory, colored according to its cluster status (top panel) or instantaneous diffusion coefficient (bottom panel). h MSD curves of clustered and unclustered trajectories from the ROI displayed in (b). Each point represents the average MSD of the indicated number of trajectories. Error bars indicate the standard error of the mean (SEM). i, j NASTIC clusters of Sx1a-mEos2 comprise “confined” molecules. The entire 17,598 trajectories dataset, as visualized in (a), was selected for analysis. i K-means clustering of MSD and vector autoregression metrics for each trajectory was used to assign them into confined (orange) and unconfined (purple). j Venn diagram showing the degree to which clustered trajectories established by NASTIC are represented by confined trajectories established using vector autoregression (VAR). Source data are provided as a Source Data file.