Imaging Bacterial Infection within Live Tissue Figure 4. Imaging of bacteria and cells within opaque infected mouse corneas. Panel (a) shows NAD(P)H autofluorescence (magenta) of corneal epithelial cells and bacteria (green) detected from optically opaque (infected) mouse eyes 24 h post-scratch and inoculation. In panel (b), bacteria (green) within the stroma of the cornea were found to orient themselves along the pattern of the collagen fibrils. Field size: 189 mm 6 189 mm. doi:10.1371/journal.pone.0024008.g004 Use of the reflection method in combination with NAD(P)H autofluorescence allowed the impact of 8 h of bacterial challenge on cell viability to be examined (Figure 6C and Video S9). That data showed that the apparent ‘‘thinning’’ of the epithelial cell layer was actually due to loss of metabolic activity of many of the surface cells; i.e. some cells were visible by the reflection method, but not by NAD(P)H AF. While there were some bacteria located at the interface between non-active (visible by reflection) and metabolically active (visible by NAD(P)H autofluorescence), bacteria were also found located between and below metabolically active cells, suggesting that bacterial traversal was not simply a consequence of cell death, despite the noted changes to cell viability. Since blotted MyD88 knockout corneas showed increased susceptibility to bacterial adhesion and bacterial traversal compared to wild-type, we next explored its impact in the absence of blotting. Surprisingly, the corneal epithelium of freshly excised MyD88 mutant eyeballs was susceptible to both bacterial adhesion (5752 6 2601 cfu/mm2) and subsequent bacterial traversal (Figure 7A, 7C) without the need for blotting, or any other form of manipulation. In contrast, bacteria only occasionally adhered to wild-type control mouse corneas (672 6 319 cfu/mm2) under the same circumstances (Figure 7B, 7C). A potential mechanism for MyD88 knockout corneal suscepti- bility to bacterial adhesion and subsequent traversal would be if the lack of MyD88 disrupted epithelial junctional integrity. To explore that possibility, MyD88 knockout and wild-type mice were compared for corneal epithelial susceptibiity to fluorescein staining/penetration, a test used widely in epithelial cell culture, Figure 5. Quantitative localization of bacteria within ex vivo blotted and bacterially challenged MyD88 deficient mouse corneas at 8 h time-point. Panel (a) shows a schematic of the custom bacterial quantification program. Each image stack is divided into ,1000 sub-volumes; in each sub-volume, the top and bottom of epithelium were localized in which 10 bins along the z-depth were assigned, followed by bacterial fluorescence quantification in each bin, and finally summation of all bins of the same z-depth across the entire image stack. Panel (b) shows quantitative analysis of bacteria traversing to the basal lamina for MyD88 (2/2) but not wild-type mice (0.00 represents the corneal surface, 1.00 the basal lamina). Average 6 S.E. of three fields are shown. doi:10.1371/journal.pone.0024008.g005 PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e24008