FIGURE 3:

Synthetic testing shows accuracy across a range of diffusivities and imaging speeds. (A) Diagram illustrating the cylindrical domain used to generate synthetic testing videos. The cylinder represents a small segment of a hypha where there is a sharp jump in diffusivity. The diameter was chosen to match the experimental z-stack distance. The purpose of the test is to quantitatively measure the ability of the pipeline to simultaneously measure low and high diffusivity adjacent within a hypha. The plots in B–D contain testing results from medial axis diffusivity projections because all quantification in Figure 4, C–F and Figure 5, B, C, E, and F was performed on the medial axis curves. (B) Top: results of analysis of simulations in which hyphae containing GEM-like particles were created to closely mimic our real data. Half the field was comprised of low diffusivity (0.01 μm² s⁻¹) particles and half high diffusivity (0.1 μm² s⁻¹), shown as a dotted black line. The z-stack acquisition rate was set at 0.85 s per stack. The videos were then passed through the full analysis pipeline. A curve representing the average diffusivity moving along the medial axis is plotted for each simulation. Each curve shows results from simulations with a different density of particles. Bottom: the diffusivity estimated from passing ground truth tracks through the analysis pipeline (bypassing the particle tracking stage). This removes error due to particle tracking and shows purely error due to sample size and spatial averaging. (C) Diffusivity estimates obtained by processing synthetic videos of varying imaging speeds through the full pipeline. For reference, the GEMs videos were imaged 0.92 s per stack on average, and GEMs particle density varied around 0.05–0.15 μm⁻³. (D) Diffusivity estimates from particle tracks obtained by processing synthetic videos of a lower diffusivity range through the full pipeline. The z-stack acquisition rate was set at 0.85 s per stack.