Imaging Bacterial Infection within Live Tissue Figure 1. 3D imaging of mouse corneas using freshly excised mouse eyeballs. Three different techniques were used to visualize cells in all layers of the cornea. Panels (a) and (d) show two-photon excitation of cytoplasmic NAD(P)H to reveal metabolically active (live) cells in the epithelium and endothelium of the cornea by autofluorescence (magenta). Panels (b) and (e) show reflection confocal imaging (red) for localization of epithelial and endothelial cells (including live and dead cells), and collagen fibers in the stromal region (red). Panels (c) and (f) show confocal imaging of CFP- tagged cell membranes (cyan) expressed in transgenic mice. All three imaging methods enabled visualization of distinct cell layers in intact corneas of at least 150 mm thickness (i.e. mouse). Panel (g) shows two-photon autofluorescence and reflection images overlaid to distinguish live and dead cells. Panel (h) shows that 3D images of CFP-membranes exhibit a high signal-to-background ratio providing excellent contrast for bacterial localization experiments. Field (xy) size: 189 mm x 189 mm. doi:10.1371/journal.pone.0024008.g001 including various innate defense responses [9]. Thus, the mechanisms by which EGTA promotes susceptibility to epithelial traversal by adherent bacteria is yet to be determined. Toll-like receptors (TLRs) are a class of receptors that bind microbial ligands (e.g. Microbial-Associated Molecular Patterns or MAMPS) or damage-associated molecular patterns (DAMPS) to trigger innate immune responses in multiple cell types including epithelia [10–12]. While it is known that some factors made in response to TLR-mediated signaling have direct antimicrobial activities (e.g. defensins) [5,13,14], the role of these, or other factors, in limiting microbial traversal through tissue epithelia has not been directly explored using actual tissue models. Here, we applied a suite of methods that together enable the position of bacteria within freshly excised, unfixed and unpro- cessed tissue to be readily visualized and quantified in 3D, and as a function of time. Using these methods, we found that MyD88, a central adaptor protein for TLR and IL-1R-dependent signaling critical in pathogen recognition, and subsequent innate immune defense responses [15], is required for epithelial resistance to both adhesion and traversal by the Gram-negative bacterium P. aeruginosa. These data suggest that epithelial defense against colonization during health involves innate defense responses that are known to be regulated. Further, the methods and results outlined in this report advance the range of tools available for studying microbial traversal of epithelia and the host factors that normally protect against it. Results and Discussion Localization of bacteria within the epithelium of fresh whole tissue The tissue we chose to use for this study, the corneal epithelium, is a multilayer of epithelial cells that covers the cornea of the eye. The cornea lends itself to imaging because it is both external and transparent. Epithelial cells on the corneal surface are viable (non- keratinized), and are normally bathed in fluid (tears) allowing the use of a water immersion objective with minimal disruption to normal physiology. The healthy corneal surface does not normally bind bacteria or other microbes. Studying bacterial interactions with the corneal epithelium thus necessitates some form of manipulation to enable bacteria to adhere. As discussed above, superficial blotting with tissue paper can be used for that purpose, and it is minimally disruptive to tissue architecture [16]. Subsequent epithelial PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e24008