Fig. 2. Time-lapse fluorescence imaging of Akt-PH-GFP recruitment to T. cruzi-associated cell membranes. Transfected L6E9 myoblasts on 25 mm coverslips were placed in a heated chamber (at 37°C) on an inverted fluorescence microscope. Akt-PH-GFP-expressing cells were located and images acquired at 5-second intervals before (0 minutes) and for 17 minutes following addition of T. cruzi trypomastigotes (2.5x10⁷ in Ringers/BSA). At 3.75 minutes post-addition, a cell-associated extracellular trypomastigote is seen as a shadow over the cell (arrow) to which Akt-PH-GFP is slowly recruited (5-9.6 minutes) and then gradually dissipates (10-17 minutes). A second cell-associated parasite, first observed at 9.6 minutes, associates with Akt-PH-GFP-enriched membrane ruffles (arrowheads) at 10.5-12.25 minutes that appear to envelope the parasite (12.8-16.3 minutes). Scale bar: 10 µm.