Extended Data Fig. 3: Single-cell motility analysis in agar by confocal microscopy.
From: Chemotaxis as a navigation strategy to boost range expansion
Thirty-second videos allowing us to track the movement of single cells were acquired (see Supplementary Video 7 for an example). a, Example of trajectories derived from cell tracking analysis. Each colour indicates the trajectory of one cell over a span of on average 75 frames (5.1 s). b, Diffusive behaviour was obtained by a linear fit of displacement variance over time (var(Δx) = 2DΔt). This analysis was performed for strain HE274 (wild type) growing in 40 mM glycerol and 100 μM aspartate (reference condition; Supplementary Text 1.5). Data shown here are for measurements in front of the expanding population (ahead of the density peak; however, the diffusion coefficient obtained at different locations does not exhibit much positional dependency, see below). Repeat of experiment showed similar results. c, Similar effective diffusion coefficients for swimming in soft-agar were obtained for other growth conditions (orange symbols; Supplementary Table 6) following the same trend as predicted from liquid culture measurements (black symbols, same as in Extended Data Fig. 2g). The diffusion measurements in soft agar were repeated twice with similar results. The data points represent means of two biological replicates. See Supplementary Table 6 for data values and conditions. d, To resolve cellular swimming behaviour of the expanding population at different spatial positions in the agar plate, videos allowing us to track single cells were acquired sequentially at a fixed position (of the agar plate) over time, for different acquisition times tacq over which videos were taken (up to several hours for each position). Image direction x was aligned with direction of migration. In this setup, the migrating population (with speed u) passes the point of acquisition at a determined time, allowing us to determine the local drift speeds and diffusion coefficients relative to the front position: x = x0 − utacq (Supplementary Text 1.5). e, Density obtained by cell counting (green line) compared to population density obtained using the approach in Extended Data Fig. 4 (fluorescence scans, red line). The spatial resolution of the latter is much coarser, each measurement point being a black dot on the red line. For comparison, the simulation result (GM model, Fig. 3) is shown in green and moderately deviates from the measured profile. f, Analysis of average displacement along x (direction of migration) and y (direction perpendicular to migration) over time for an acquisition time tacq corresponding to a position at the front bulge (x = 21.3 cm, indicated by the dashed lines in e, g, h). The average displacement (purple symbols) increased linearly in time along the direction of migration but was negligible perpendicular to the direction of migration (fitted purple lines show drift speed in each direction, ⟨Δx/Δt⟩ and ⟨Δy/Δt⟩). g, Position dependence of the drift (in the direction of expansion) was determined at different tacq, corresponding to different positions of the expanding population. For ease of reference, cellular densities (e) are shown again as green symbols. Up to the resolution of the data, the drift velocity vanished to the left of the density trough (x < 19 mm). h, Position dependence of the diffusion coefficient. Using the approach from b to determine the diffusion coefficient at different tacq, we obtained the results shown as orange symbols. A moderate (~20%) increase in D is observed at the very front of the population. This spatial dependence may be due to the accumulation of faster swimming cells at the front9. All data in e–h are from a single expansion experiment done under reference conditions (40 mM glycerol + 100 μM aspartate; 2:1 mixture of fluorescent variant HE274 and non-fluorescent variant HE339). Similar results were obtained for one biological replicate. Error bars in e, h denote s.d. and were calculated from repeated observations at three different times during the same expansion process.
