Imaging Bacterial Infection within Live Tissue Figure 9. Impact of MyD88 deficiency on colonization in vivo. Similar to the ex vivo results at the same time-point, panels (a) and (b) show blotted corneas of MyD88 (2/2) mice were more susceptible to both bacterial adherence and traversal after 8 h of bacterial challenge as compared to blotted wild-type which showed bacterial adherence only. This observation was confirmed by quantitative localization of GFP bacteria using the custom program as shown in panel (c). Whereas in the absence of blotting, panels (d) and (e) show bacterial traversal in vivo was disabled. Only bacteria adherence was detected on MyD88 (2/2) (d) but not wild-type (e) mouse corneas. This observation was confirmed by quantitative localization of GFP bacteria using the custom program as shown in panel (f). doi:10.1371/journal.pone.0024008.g009 include antimicrobial peptides, or infiltrating phagocytes (in vivo) [38–43]. Junctional integrity has also been shown to be responsive to TLR-, and MyD88-, mediated signaling in intestinal epithelia [44]. While the data in Fig. 9 show normal barrier function in na¨ıve MyD88 knockout mice (and therefore that tight functions are functionally intact), it remains possible that regulation of junctional integrity in the face of bacterial challenge is compro- mised in the corneas of MyD88 knockout animals. Other possibilities are that mucins (or other epithelial surface-associated moieties that either bind or repel bacteria) are dependent on MyD88, or that they act in synergy with MyD88-mediated factors (such as antimicrobials) to exert their protective effects. Further research will be needed to sort through these and other potential possibilities for MyD88 involvement in epithelial defense against traversal by microbes. Conclusion In this report, we demonstrate that the combination of various imaging methods can be used to further our understanding of bacterial-host cell interactions in live unprocessed tissues. These relatively simple and robust non-invasive methods, used with strategies for manipulating tissue susceptibility to microbes, and a custom data analysis program, represent a set of novel, quantitative tools for studying both pathogenesis of, and defenses against, infection. Using them, we have shown directly that MyD88 plays a critical role in defense against epithelial colonization by a Gram-negative opportunistic pathogen, and demonstrating that host-pathogen interactions reported to occur in vitro (e.g. membrane bleb-niche formation) can also occur when tissues are challenged intravitally. The data presented also suggest new insights into bacterial pathogenesis not previously reported (such as a spatio-temporal relationship between bacteria and collagen fibrils during infection), that can now be expanded upon using the developed methods. While there are clear advantages to using mice, including the availability of gene knockouts and other reagents, these methods could be used with eyes from other species or with other types of tissue. With the correct combination of resources, and if image stabilization can be achieved at the high magnification needed to detect infecting bacteria, these methods could be implemented in vivo using anesthetized live animals as has been done for studying immune cell trafficking [45]. Since these imaging methods are non-invasive, there could even be potential for translation to use in humans. Materials and Methods Ethics Statement All procedures involving animals were performed in strict accordance with a protocol approved by the Animal Care and Use Committee, University of California, Berkeley, which is an AAALAC accredited institution. Protocol #R203-1210B. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e24008