Figure S4. Tracking diffusing particles is compromised at high particle densities.

(A) Results of a simulation where the indicated number of INV-sized particles was diffusing with known D in a 3D space and captured in a confocal plane. Simulated images were captured at 10-, 60-, and 100-ms intervals and then particles were tracked using TrackMate (v7.10.2). At 60 ms, the frame rate used in this study, tracking is close to the ground truth value at D < 0.1 and degrades for particles with higher coefficients. Higher densities lead to greater underestimation as tracks become mislinked because of particle density. Slower frame rate capture leads to consistent underestimation of D. (B, C, D, E) Bleaching of StayGold-TPD54 to reduce the number of INVs to be tracked leads to more accurate estimation of D. (B) Stills from a region of a cell expressing StayGold-TPD54 before (pre) and after (post) bleach. (C) Tracking in such regions leads to a decrease in the number of particles to track. Median track density is the number of neighboring tracks in a 1-μm radius circle. So “pre” equates to 5.57 vesicles and “post” to 2.55 vesicles μm⁻². (A) These densities translate to respective values of 16,982 and 7,774 vesicles for the simulation shown in (A). (D) Diffusion coefficient measured in the same cells pre- and post-bleach. (E) Mean squared displacement for these datasets.