traversal by the adherent bacteria can then be toggled on (or off), by use (or omission) of EGTA treatment after the blotting procedure. With methods in hand to encourage bacteria to interact with the epithelium, the next goal was to develop methods for visualizing/ localizing bacteria within the bacterially-challenged tissue that do not require processing (i.e. fixation, labeling, staining) of the tissue. Details of the methods utilized are outlined in the materials and methods. Briefly, bacteria were visualized using a multicopy plasmid expressing enhanced green fluorescent protein (GFP). Corneal epithelial cells within bacterially challenged, and then freshly excised unprocessed eyeballs, were imaged using one or more of three different techniques: 1) NAD(P)H autofluroescence (AF) (multiphoton, images cytoplasm of metabolically active cells). 2) A reflection technique (confocal, images both live and dead cells). 3) Use of transgenic mice expressing membrane-bound cyan fluorescent protein (CFP) (confocal, enables cell membranes to be visualized). All three methods enabled cells within all layers of the cornea (epithelium, stroma and endothelium) to be visualized (Figures 1A, B, C). Thus, it was possible to accurately localize the upper and lower limits of each layer, providing landmarks for quantifying the depth of microbe penetration using any of the three methods. Each of the three imaging modalities had unique features for gaining more detailed information. For example, since NAD(P)H resides within the cytoplasm, the NAD(P)H AF method enables subcellular imaging; the cytoplasm fluoresces, while the nucleus and plasma membrane appear black in contrast (Figure 1D). Since its fluorescence intensity is proportional to cellular metabolic activity [17–19], this technique also provides information about the health of individual cells (Figure 1D). The reflection confocal and membrane-CFP methods each allow positive (rather than negative) visualization of epithelial cell boundaries, and thus can be used to ensure that both dead and live cells are accounted for (Figures 1E, F). Thus, when used in combination with NAD(P)H AF, confocal or membrane-CFP can enable live and dead cells to be distinguished from one another (e.g. Figure 1G). The AF and reflection methods (in contrast to membrane CFP) do not require a specific type of genetically modified animal, which means that any animal can be studied, including wild-type and all genetically modified species. Membrane-bound CFP, on the other hand, provides a high signal-to-background ratio, and consequently a high image contrast of cell membranes (Figures 1F, 1H and Video S1), compared to either AF or reflection (Figures 1D, E respectively), making this an ideal method for determining if infecting bacteria are intracellular or extracellular. Images from bacterially-challenged corneas (6 h) are shown in Figure 2. The top panels show results for corneas that were first blotted and then treated with EGTA for 1 h prior to bacterial challenge. Both the confocal CFP membrane method (Figure 2A and Video S2) and the NAD(P)H autoflurescence method (Figure 2B and Video S3) revealed epithelial traveral by the bacteria; i.e. many bacteria were detected deep within the multilayered corneal epithelium. Consistent with our previously published findings showing that the the basal lamina between the epithelium and the stroma functions as a barrier to bacterial passage [20], few bacteria were noted in the underlying stroma. Also consistant with our previous results [16], corneas that were not subsequently treated with EGTA prior to bacterial challenge (blotted only, bottom panels and Video S4) were susceptible to bacterial adhesion only, without bacterial penetration beyond the epithelial surface. Having established methods for enabling and also visualizing bacteria deep within unprocessed and unsectioned live tissue, we Imaging Bacterial Infection within Live Tissue next explored if these methods could be used to provide more detailed spatial and temporal information about the traversal process and its impact. Use of the CFP membrane mouse and high magnification enabled the position of bacteria relative to cell membranes, and the status of those membranes, to be determined. For example, perusal of images in Figures 2 reveals that the CFP signal is relatively diffuse after bacterial traversal of blotted/ EGTA-treated corneas relative to the very clearly localized CFP signal seen in the uninfected eye in Figure 1. Blotting/EGTA treatment used alone without bacterial challenge did not cause CFP diffusion (data not shown), suggesting that it was the bacterial traversal per se that impacted the appearance of the cell membrane marker; perhaps not surprising considering that P. aeruginosa can elaborate multiple toxins [21] and basolateral cell surfaces are highly susceptible to them [22]. Use of the CFP-membrane mouse also revealed that P. aeruginosa can induce the formation of membrane blebs, and can localize within them, a phenomenon which we have reported to occur when P. aeruginosa infects cultured corneal epithelial cells [23,24]. Figures 3A and 3B each show examples of membrane blebs; the higher magnification used in Figure 3B enabled a bacterium to be located within a bleb. While the significance of ‘‘bleb niche’’ formation in epithelial cells during P. aeruginosa infection is still under investigation, this finding that they occur in actual infected fresh tissue, and not only in cells grown and infected in tissue culture wells, lends justification to that effort. One clear advantage of imaging live versus fixed tissues is that temporal information can be obtained using the same sample. For example, individual bacteria and cells within the epithelium of fresh tissue can be tracked over time for their location and/or their viability (Figures 3B, 3C and Video S5). Video S5 shows that the bacterium located within the membrane bleb in Figure 3B is exhibiting swimming motility inside the bleb, confirming another phenomenon that we have reported with cultured cells, but this time for a cell located within the corneal epithelium of a live eyeball. Figure 3C (high magnification z -stack) shows several bacteria in the basal (deep) layers of the corneal epithelium of intact eyeballs mostly located between epithelial cells. In this image moving bacteria are shown in red, while white bacteria are stationary since they were captured twice (sequentially in CFP and GFP channels) in the same z-plane (temporally spaced images were overlaid). In addition to providing insights into the temporal details of bacteria-cell interactions, these methods could also be used to monitor individual host cell responses to bacterial challenge over time (e.g. by morphology or by tracking NAD(P)H AF), and with respect to the position of individual bacteria. Lack of significant bacterial penetration into the stroma as a result of an intact basal lamina likely explains why blotting/EGTA treatment is not sufficient to enable susceptibility to overt (clinically visible) disease despite bacterial traversal through the epithelium [16]. We next explored the usefulness of these imaging methods when there is overt disease, i.e. when the cornea becomes optically ‘‘opaque’’ (non-transparent) due to the infiltration of leukocytes, e.g. neutrophils and monocytes [25–27]. Disease was induced by scratching the cornea through to the level of the anterior stroma (damages basal lamina barrier) prior to challeng- ing with bacteria [28,29]. It remained possible to detect cellular NAD(P)H AF and to localize GFP-labeled P. aeruginosa, even when the infecting bacteria were located deep within the stromal region of these opaque corneas (Figure 4, Video S6). The images collected revealed interesting insights illustrating the power of this technology compared to fixed/sectioned tissue. For instance, the images shown in Figures 4A, 4B and Video S6 were collected at a region of the cornea where the basal lamina was not directly PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e24008