Source of Mice MyD88 (2/2) mice fully backcrossed to a C57BL/6 back- ground were provided by Dr. Greg Barton (University of California, Berkeley, CA). C57BL/6 transgenic mice expressing membrane-bound CFP under the control of human ubiquitin C promoter were also used in some experiments [46]. Age-matched C57BL/6 and Black Swiss wild-type mice were purchased from Taconic Farm Inc. (Hudson, NY). Bacterial strain and inoculum preparation Pseudomonas aeruginosa invasive strain PAO1 expressing enhanced GFP (PAO1-GFP) from a highly stable, multicopy plasmid (pSMC2) was used [47] (kindly provided by Dr. Gerald B. Pier, Harvard Medical School). Plasmid transformation of bacteria was achieved by standard electroporation. Bacteria were grown on tryptic soy agar plates supplemented with carbenicillin 300 mg/ml at 37uC for ,16 h. Inocula were prepared by suspending bacteria in Dulbecco’s Modified Eagle’s Medium (Lonza, Walkersville, MD) to a concentration of ,108 or 1011 cfu/ml. We have previously confirmed that the bacteria can retain this, and other, plasmids in vivo even without antibiotic selection (up to 48 h and longer) [48,49], and show strong fluorescence using conventional and confocal fluorescence microscopy. Ex vivo traversal model C57BL/6 wild-type and MyD88 (2/2) gene knockout mice were subject to lethal injection with anesthesia cocktail (21 mg/ml ketamine, 2.4 mg/ml xylazine, and 0.3 mg/ml acepromazine). Whole eyes were enucleated to exclude tear fluid before a PBS rinse (,10 ml), tissue paper blotting (when used) with KimwipeTM (Kimberly-Clark) and incubation with 200 mL of bacterial suspension (,1011 cfu/ml) at 35uC for 4 or 8 h before imaging. In some experiments, blotted eyeballs from transgenic CFP- membrane mice and Black Swiss wild-type mice were treated with EGTA (100 mM in PBS) or PBS alone for 1 h before bacterial incubation for 6 h (both treatments at 35uC). In vivo traversal model C57BL/6 wild-type and MyD88 (2/2) gene knockout mice were subject to anesthesia by intraperitoneal injection (50 ml/25 g of body weight) of ketamine/xylazine/acepromazine cocktail. One cornea of each of the anesthetized mice were rinsed with PBS to wash away tear fluid, then blotted with KimwipeTM tissue paper (when used) and inoculated with 5 ml of bacterial suspension (,1011 cfu/ml). Animals were euthanized 8 h post-inoculation by lethal injection, and eyes were carefully enucleated, then rinsed with PBS (,10 ml) prior to imaging. Scratch infection model One cornea of anesthetized mice was scratched linearly three times using a sterile 25-gauge needle, then inoculated with 5 ml of bacterial suspension (,108 cfu/ml). Animals were euthanized 24 h post-inoculation when corneal opacity had become obvious. Eyes were carefully enucleated and rinsed with PBS prior to imaging. Technologies for visualization of bacteria and host cells GFP-expressing bacteria were excited by the 488 nm argon ion laser line (different fluorophores, e.g. RFP, can be used for bacteria as needed) or the multiphoton laser at 920 nm (in opaque mouse corneas). To visualize corneal epithelial cells in live unprocessed eyeballs, a combination of previously described two-photon and confocal imaging techniques were used [50,51]: (1) Multiphoton Imaging Bacterial Infection within Live Tissue excitation at 720 nm from the Ti:Sa laser with a pulse width of ,100 fs and maximum average power of ,1.6 W into the microscope, which excites endogenous NAD(P)H molecules in metabolically active living cells [17–19]. Corneas were previously used to show the potential of in situ NAD(P)H autofluroescence (AF) via two-photon microscopy for assessing cellular metabolic states [17]. (2) HeNe laser line at 633 nm, which allows imaging of both live and dead cells by detecting reflected light [51]. (3) Argon ion laser line at 458 nm, which excites membrane-bound CFP expressed in transgenic mice. Combined two-photon/confocal imaging of whole eyeballs Mouse eyeballs examined for bacterial traversal were rinsed with PBS three times to remove non-adherent bacteria. After rinsing, the back of an eyeball (where the optic nerve is located) was fixed on a 12 mm round glass coverslip with cyanoacrylate glue to maintain an upright position (i.e. cornea facing up). The coverslip/eyeball was placed in a 47 mm Petri dish which was then filled with Ham’s F-12 medium (Lonza, Walkersville, MD) to cover the eyeball completely, and was ready for imaging using a 63x/0.95 water-dipping objective of an upright two-photon/ confocal microscope (LSM 510 META NLO Axio Imager; Carl Zeiss, Thornwood, NY) equipped with a tunable, ultrafast and dispersion-compensated Ti:Sapphire laser (Mai Tai DeepSee; Spectra-Physics, Santa Clara, CA). A 435–485 nm bandpass or a 500–550 nm bandpass filter was used to detect NAD(P)H AF or GFP respectively. Three or more random fields of each eye were imaged from the corneal surface through the entire epithelium in 0.5 mm steps. Three dimensional images were reconstructed from z-stacks using ImageJ (NIH). Computer program for quantitative localization of bacteria Data analysis was performed with IDL (ITT Corp., Boulder, CO; code available upon request). The epithelium was divided by binning adjacent x-y pixels into typically 1.48 mm x 23.75 mm. Sub-division of the image stack and analysis of the fluorescence profiles as a function of z to locate the apical and basal sides of the epithelium was found to be superior to Sobel and Roberts edge enhancement filters which did not give satisfactory results. For each sub-volume, we had two relevant channels: one containing the structural information regarding the epithelia (AF, reflection, or CFP), and the other containing bacterial fluorescence (e.g. GFP). AF and CFP give a similar profile, characterized by a single central peak in fluorescence as a function of z, while reflection requires a slightly different strategy to locate the apical/ basal surfaces. After sub-division, the AF or CFP data stack was optionally smoothed, depending on the signal-to-noise ratio, using a boxcar average. The central peak was then located using a peak- finding algorithm which computes the derivative of the fluores- cence trace by convolution with a Savitsky-Golay filter. The order and number of points of the filter were selected by the user. The peak was located by searching for zero crossings in the derivative. Once located, this point marked the center of the epithelium. At this point, the apical and basal edges of the epithelium were located by using the same peak-finding algorithm to locate the peak values of the derivative of the AF or CFP profile from the beginning of the stack to the center of the epithelium (apical) and from the center of the epithelium to the end of the stack (basal). When using reflection to locate the surfaces the situation is slightly different. Reflection profiles have two strong peaks: one at the surface of the cornea and one at the basal lamina. Hence, the PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e24008