Figure 1: Single-molecule tracking (SMT) methodology.

U2OS cells stably expressing an EGFP-tagged N-terminal fragment of Lamin B Receptor (LBR) to mark the nuclear periphery are grown on glass bottom chamber slides. Once at an optimal confluency, cells are transiently transfected with a Halo-tagged protein of interest. After 24 h of transfection, 1 nM of Halotag ligand (JF646) is added for 30 min followed by a series of washes to remove unbound ligand before imaging. Highly inclined and laminated optical light sheet illumination (HILO) is employed to image a thin optical section and limit out-of-focus fluorescence. A series of images are taken in the same plane using simultaneous 488 nm and 647 nm excitation with a 30 ms acquisition time every 200 ms. Following acquisition, imaging channels are split using Image J and imported into a customized MATLAB software to annotate a region of interest (i.e. LBR fragment localization), individual spots are then tracked over time. Single-molecule tracks collected amongst different cells and experiments are aggregated, undergo photobleaching correction, and are fit to a one-, bi-, or multi-component exponential distribution.