Fig. 2.

Benchmarking two-photon data analysis methods. The power of two-photon data analysis methods can be evaluated systematically using computer-generated data (Materials and Methods) as shown here for the three methods discussed in this paper. (A) Naive T cell data (red; 1,132 tracks) are displayed for comparison alongside three simulated datasets: random walk (black; 842 tracks), random walk with taxis (blue; 3 μm/min along z axis, 897 tracks), and a mixed population with 50% taxing cells and 50% randomly walking cells (green; 5.1 μm/min along z axis, 893 tracks). Simulated cells were tracked in a finite volume having the size of our two-photon imaging region. (B) Mean square displacement of the data in A plotted as a function of time. (C) Cell steps extracted from the tracks in A. Crosses indicate the null vector (red = 703 steps; black = 693 steps; blue = 773 steps; green = 749 steps). (D) Analysis of the mean angle between the cell steps (C) and the taxis direction, which is known only for the simulated data. A mean angle of less than 90° indicates taxis (mean angles and 95% confidence intervals are shown; asterisk indicates significance). (E) Hotelling's T² test applied to the y and z dimensions (compare with Fig. 1D).