<i>Toxoplasma</i> egress from VERO cells in 3D collagen matrix.

A. Microscopy images at 20x of a VERO cell infected with RFP-expressing Toxoplasma (RFP-Tg) for 60 h in a 3D collagen matrix (XY view), showing a representative image of the same PV just before (panel a) or after rupture with parasites escaping in various directions (panel b). Left: phase-contrast images; right: fluorescence microcopy. Arrowhead shows the infected cell from where the parasites escaped. Bar, 40 μm. B. Live fluorescence microscopy of a VERO cell multi-infected with RFP-expressing Toxoplasma, in a 3D collagen matrix, at the indicated times p.i. Images show top-down (XY) and side-on (YZ) views of the infected VERO cells over the course of parasite egress. After 52 h of infection, individual parasites escape the ruptured cell in all directions and at 60 h p.i. a more diffuse fluorescence signal appears concomitant with parasite dispersion in the matrix as they move away from the former host cell. Scale bars, 50 μm. C. Quantification of fluorescence intensity of RFP-expressing Toxoplasma within the PV (48 h p.i.) or during egress (60 h p.i.). Regions-of-Interest corresponding to the fluorescence signal of parasites were drawn at the indicated distances from the ruptured cell to collect numbers that are integrated pixel intensity of entire area minus background using the ImageJ analysis program. Data are means of three independent biological experiments (n = 4 to 9 PV) showing statistical differences between distances from the PV centroid at 48 and 60 hpi (*, p < 0.05 with two-tailed t-test).