Imaging Bacterial Infection within Live Tissue Figure 8. MyD88 corneas do not label with fluorescein suggesting tight junctions are intact ex vivo and in vivo. Fluorescein was added to tissue paper blotted or normal healthy (non-blotted) mouse corneas to access cell-cell junction integrity. Extent of corneal staining was examined using a slit lamp (a-c) and confocal microscopy (d–i). Panels (a, d, g) show extensive fluorescein staining (green) in blotted wild-type (C57BL/6) corneas, but not non-blotted wild-type (b, e, h) or MyD88 (2/2) (c, f, i) mouse corneas. Z-stack confocal images (1272 mm 6 1272 mm 6 100 mm) are presented as 3D block view (d, top), 3D orthoslice view (d, bottom), and 2D x-y view (g) for blotted and fluorescein stained wild-type mouse cornea. In parallel, 3D block views and 2D x-y views of confocal images are shown for normal wild-type (e, h) and MyD88 (2/2) (f, i) mouse corneas. These data show that epithelial tight junctions are intact in MyD88 knockout corneas prior to bacterial exposure. doi:10.1371/journal.pone.0024008.g008 vitro study we showed that tear fluid can modulate epithelial immunity directly to protect cells against various bacterial virulence strategies, involving upregulation of both RNase7 and ST2 [37]. Thus, we performed experiments in which the blotting and bacterial challenge steps were both performed while the eye was in vivo. Experiments were done both with and without blotting using 8 h of bacterial challenge, and both wild-type (control) and MyD88 knockout mice. With blotting, the in vivo results mirrored the ex vivo results, i.e. both wild-type and MyD88 knockout mouse corneas bound bacteria, with more extensive binding and also epithelial traversal by bacteria for the MyD88 mutant eyes (Figure 9A–C). As expected, bacteria did not associate at all with non-blotted wild-type corneas in vivo (Figure 9E, 9F). MyD88- deficient corneas showed only low level bacterial adhesion and only shallow bacterial penetration beyond the surface (Figure 9D, 9F). This contrasted with results obtained for experiments done ex vivo that yielded significant adhesion and penetration to the basal lamina. Indeed, ex vivo data showed maximal GFP fluorescence intensity was 12,000 at a depth of 0.4 towards the basal lamina (Figure 7); in vivo it peaked at only 1,200 and at a more shallow level of 0.2 (Figure 9F). Thus, the unmanipulated MyD88-deficient corneas were less susceptible to bacterial colonization in vivo, than they were when removed and challenged in vitro. Whether this is explained by (MyD88-independent) protective biochemical factors in vivo, or simply by physical removal of bacteria during blinking/ tear flow, is yet to be established. Blotting/EGTA pretreatment versus MyD88 knockout mice as models for enabling traversal to be studied While useful for studying defenses against traversal, these models also provide systems that could be used to enable the traversal process to be studied (e.g. identifying bacterial factors involved). The two approaches have different advantages; i.e. blotting/EGTA treatment can be used with mice of any background (i.e. wild-type, gene knockouts, or transgenic mice such as the CFP membrane mice), while use of MyD88 knockouts does not necessitate chemical or other treatment of the tissue to enable traveral. Given the known functions of MyD88, its role in defense against both bacterial adhesion and bacterial traversal are likely to involve either TLR- or IL-1-mediated epithelial-, or resident macrophage or dendritic cell-, derived/initiated innate defenses. These could PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e24008