Imaging Bacterial Infection within Live Tissue Figure 3. Temporal and spatial tracking of EGTA-enabled bacterial traversal. Traversal was enabled in the corneal epithelium of transgenic mice expressing CFP-tagged cell membranes (cyan). Panels (a, b) show bacterial-induced membrane bleb formation ex vivo visualized as spherical membrane projections (arrow) extending away from the epithelial cells. A representative view in the xz plane is shown in (a), and a higher magnification imaging revealing a bleb-confined bacterium is shown in (b). In (c), bacteria can be seen located between cells (orange arrows) where some were motile (fast-moving GFP-bacteria, red; slower-moving GFP-bacteria, white = captured twice in both CFP and GFP channels). Other bacteria in this image appeared to be in the cytoplasm (red arrow). doi:10.1371/journal.pone.0024008.g003 deficiency was found to be similar to EGTA treatment, i.e. it enabled bacteria to traverse deeply through the blotted, multilay- ered corneal epithelium with a significant number reaching the basal lamina by 8 h post-inoculation as compared to wild-type controls that showed no significant traversal (Figure 6A and Video S7). Close inspection of the NAD(P)H AF images suggested that the epithelium of MyD88 knockout murine corneas was altered after the blotting and the subsequent 8 h of bacterial challenge. Many of the epithelial cells were rounded, and the total number of cell layers appeared reduced. While cell rounding was also seen after bacteria had traversed EGTA-treated corneas (Figures 2 and 3), the apparent ‘‘thinning’’ of the entire epithelial layer was observed only for challenged MyD88 knockout eyes. To explore the mechanism for these morphological changes, experiments were repeated using a shorter challenge time frame (4 h versus 8 h). The data showed unusually large numbers of bacteria adhering to the surface of MyD88 knockout corneas at 4 h, without significant traversal (Figure 6B and Video S8). MyD88 mutation enhanced bacterial adhesion at time points earlier than it enabled traversal. Further, the underlying corneal epithelium appeared morpholog- ically normal at the earlier (4 h) time point. Thus, changes to epithelial morphology noted at 8 h must have occurred after bacteria bound, and were probably caused by the bacteria, rather being a direct result of MyD88 mutation. Indeed, we might expect cells lacking MyD88 (important in innate defense responses) to be more sensitive to negative effects of bacteria than ‘‘normal’’ EGTA exposed cells. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e24008