Reconstruction of contact guidance of migrating T cell by collagen fibers. (A) The outline of the cell depicted in Figure 1 was obtained every 24 seconds and superimposed on the 3D reconstructed backscatter signal of the collagen fibers. Cell boundary (blue), 3D collagen matrix (green; 10 μm in depth), and colocalization of cell boundary and individual collagen fibers (cyan, arrowheads). The mean path and pseudopodal extensions are shown by the solid red line. Migration occurred toward the top right corner (black arrow). (B) False color representation of the physics of the transmigrated path (white segments), calculated from pixel colocalization between T-cell boundary and collagen fibers. (C) Extracted cell boundary (gray line) and segments colocalized with collagen fibers (black sections). Asterisks indicate turns guided by outside fiber cues. (D) Definition of 4 virtual tracks along the migration path to approximate 4 major morphological regions of a polarized T cell; for instance, lateral portions of the anterior head (a, d) and posterior cell parts near the uropod (b, c). (E) Calculation of pixel series that represent black segments in (C) for each track along the migration path (E, top graph), resulting in the cumulative number of fibers that were simultaneously aligned along the forward moving cell edge (E, bottom graph). These reconstruction data are representative for more than 5 independent cells reconstructed by dynamic confocal backscatter imaging (compare movies 1-3). Bars, 5 μm.