Imaging Bacterial Infection within Live Tissue Figure 2. 3D imaging of bacterially challenged eyeballs. All eyeballs were tissue paper blotted to enable bacterial adherence to the corneal epithelium. Susceptibility to traversal by adherent bacteria was then toggled on/off by use/omission of EGTA treatment following the blotting procedure. Upper images (EGTA used to toggle on traversal) show deep bacterial traversal 6 h after bacterial challenge in EGTA-treated corneas. Lower images were PBS-treated controls (EGTA step omitted) and showed bacterial adherence to the surface without subsequent bacterial traversal. Panel (a) shows confocal technique using mice with CFP-tagged cell membranes (cyan), and panel (b) shows two-photon images of cellular NAD(P)H autofluorescence (magenta) in transgenic (C57BL/6 background) or wild-type (Black Swiss background) mouse corneas respectively. Field size: 189 mm 6 189 mm. doi:10.1371/journal.pone.0024008.g002 impacted by the scratching process. This z-stack was collected with the optical axis of the microscope parallel to, but laterally shifted from, the optical axis of the eye and as a result of the curvature of the eye, the image captured in Figure 4A (taken from the stack) shows the epithelium at the top and the interface between the epithelium and the stroma (basal lamina region) towards the bottom. This enabled direct visualization of the ‘‘filtering’’ effect of the basal lamina, i.e. bacteria can be seen deep in the epithelium arranged in circular patterns surrounding cells/nuclei with concentrations tapering off at, and under, the basal lamina. While the images collected directly under the basal lamina showed a distinct bacteria free zone, deeper regions of the stroma again revealed the presence of bacteria, this time arranged in a different pattern. Here, bacteria were found radiating out laterally in single file, end-to-end lines, corresponding with orientation of the stromal collagen fibrils (Figure 4B, Video S6). Images collected from adjacent regions showed that these bacterial ‘‘trains’’ originated at the scratched region where bacteria enter the corneal stroma directly through the damaged basal lamina, and then eminated away from the area in multiple directions. Whether P. aeruginosa utilizes a form of surface-associated motility along collagen fibrils to disseminate through the cornea is to be determined. This is of interest, considering that twitching motility is a critical virulence factor for P. aeruginosa in this corneal infection model [30,31]. Impact of MyD88 on bacterial traversal To gain further mechanstic insights into how the corneal epithelium normally defends itself against bacterial traversal, the role of MyD88, a central adaptor protein for TLR signaling, was examined. This was done by comparing wild-type to MyD88 knockout (2/2) mice. After image acquisition (z-stacks), image stacks were analysed using a custom program which permited quantitative z-axis profiling of bacterial traversal from any of the three signals that localized the structure of epithelium in addition to bacterial fluorescence (Figure 5A). To take tissue surface irregularities into account (the corneal surface is curved), an image stack (a field) was divided into approximately 1000 sub-volumes for analysis. In each sub-volume, the algorithm located the apical and basal sides of the epithelium. The epithelium was sub-divided into ten bins along the z-axis relative to its local thickness. Background subtraction was performed on the corresponding bacterial fluorescence trace for each sub-volume. The remaining fluorescence intensity of bacteria, which served as a proxy for the number of bacteria, in each z-bin was integrated. The sum of local profiles across all sub- volumes provided the final fluorescence intensity as a function of traversal distance for a particular field. Figure 5B shows a quantitative comparison of wild-type to MyD88 knockout mice on traversal after tissue paper blotting to allow adherence (no EGTA treatment). The impact of MyD88 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e24008