Imaging Bacterial Infection within Live Tissue Figure 6. Impact of MyD88 deficiency on defense against epithelial traversal by bacteria after blotting. Panel (a) shows two-photon images of cellular NAD(P)H autofluorescence (magenta) of MyD88 (2/2) (top) and wild-type (bottom) mouse corneas. Deep bacterial traversal (GFP, green) of corneal epithelium was detected in MyD88 (2/2) but not wild-type mice 8 h after bacterial challenge ex vivo. Only adherent bacteria without epithelial traversal were found in tissue paper-blotted wild-type corneas. Panel (b) shows an earlier (4 h) time-point at which a relatively larger number of bacteria were found adhering to the MyD88 (2/2) corneal epithelium as compared to the wild-type. In panel (c), merged image (fuchsia) of reflection (red) and autofluorescence (magenta) is shown. Reflection confocal methodology (red) can be used to capture cells not visualized by autofluorescence (i.e. dying or dead cells) to monitor cell viability during bacterial traversal. This method confirmed that penetrated bacteria at the later (8 h) time point were overlaid with epithelial cells. doi:10.1371/journal.pone.0024008.g006 whole tissues, and in the eyes of human patients in routine clinical practice, to assess epithelial permeability/junctional integrity [32– 34]. Confocal microscopy was used to determine depth of fluorescein penetration. Even without subsequent EGTA treat- ment, the epithelium of blotted wild-type mouse corneas was susceptible to deep penetration by fluorescein (Fig. 8). In contrast, neither wild-type nor MyD88 knockout corneas labeled with fluorescein when not blotted (Fig. 8). Therefore, susceptibility of unblotted MyD88 knockout corneas to bacterial adherence and traversal was not due to disruption of epithelial tight junctions prior to bacterial exposure. Further, the data suggest that tight junctions do not act in isolation to modulate bacterial traversal in wild-type eyes. Whatever the case, these two models enable traversal by different mechanisms, which will provide additional tools for subequent studies of epithelial defense against infection. Since bacteria bound to (and penetrated) unblotted MyD88 deficient corneas, MyD88 is required for defending the healthy corneal epithelial surface against bacterial adhesion, in addition to protecting it against bacterial traversal. How these findings reconcile with the established role of surface-associated mucins in protecting against bacterial adhesion is not yet clear. The above studies were done using freshly excised eyeballs. We have reported that tear fluid, which bathes the corneal epithelium in vivo, is protective against bacteria [35,36]. In a more recent in Figure 7. Impact of MyD88 deficiency in the absence of blotting. Panels (a) and (b) show two-photon images of cellular NAD(P)H autofluorescence (magenta) in non-blotted MyD88 (2/2) or wild-type mouse corneal epithelium 8 h after bacterial challenge ex vivo. Significant bacterial adherence and traversal (GFP, green) were found in MyD88 (2/2) mouse (a) but not wild-type mouse corneas (b). This observation was confirmed by quantitative localization of GFP bacteria using the custom program as shown in panel (c). doi:10.1371/journal.pone.0024008.g007 PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e24008