peak finding algorithm was used to locate these two peaks. The edges of the epithelium were located by peak-searching the derivative of the reflection signal from the beginning of the stack to the surface peak (apical) and from the basal lamina peak to the end of the stack (basal). To quantify bacterial traversal in each sub-volume, a threshold was used first to determine if bacteria were present. This threshold is set by the user and must be adjusted depending on the particular image stack. If there was significant bacterial fluorescence, the corneal thickness was binned into ten z-bins and the fluorescence is integrated over each bin. This gave a local profile of the bacterial traversal of the epithelium. The sum of the individual profiles gave an overall representation of the traversal in the image stack. Supporting Information Video S1 CFP-membrane mouse corneas were tissue paper blotted, PBS treated for 1 h, then bacteria- challenged for 6 h ex vivo before confocal imaging. Moving XZ and XY planes of the 3D image show individual corneal epithelial cells (CFP membrane, magenta) with GFP bacteria (green) adhering on the epithelial surface. (MOV) Video S2 Confocal imaging of ex vivo blotted, EGTA treated (1 h) and bacteria-challenged (6 h) CFP-membrane mouse cor- neas. Moving XZ and XY planes of the 3D image show corneal epithelial cells (CFP membrane, magenta) with GFP bacteria (green) deeply traversing the corneal epithelium to the level of the underlying basal lamina. (MOV) Video S3 Two-photon and confocal imaging of ex vivo blotted, EGTA treated (1 h) and bacteria- challenged (6 h) wild-type Black Swiss mouse corneas. Moving XZ, YZ and XY planes of the 3D image show corneal epithelial cells (cytoplasmic autofluorescence, magenta) with GFP bacteria (green) deeply traversing the corneal epithelium to the level of the underlying basal lamina. (MOV) Video S4 3D block view of the confocal image of ex vivo blotted, PBS treated (1 h) and bacteria- challenged (6 h) CFP membrane mouse corneas. GFP bacteria (green) were found to adhere to the corneal surface without traversing the epithelium. CFP cell membranes are shown in red. (MOV) Video S5 Combined two-photon and confocal time-lapse imaging of GFP bacteria (green) confined in a CFP membrane bleb (cyan) of the corneal epithelium (cytoplasmic autofluores- cence, magenta) of transgenic CFP-membrane mouse corneas blotted, EGTA treated (1 h) and bacteria-challenged (6 h) ex vivo. (MOV) References 1. Kohler H, Sakaguchi T, Hurley BP, Kase BA, Reinecker HC, et al. (2007) Salmonella enterica serovar Typhimurium regulates intercellular junction proteins and facilitates transepithelial neutrophil and bacterial passage. Am J Physiol Gastrointest Liver Physiol 293: G178–187. 2. Macutkiewicz C, Carlson G, Clark E, Dobrindt U, Roberts I, et al. (2008) Characterisation of Escherichia coli strains involved in transcytosis across gut epith- elial cells exposed to metabolic and inflammatory stress. Microbes Infect 10: 424–431. 3. Soong G, Parker D, Magargee M, Prince AS (2008) The type III toxins of Pseudomonas aeruginosa disrupt epithelial barrier function. J Bacteriol 190: 2814–2821. 4. Zasloff M (2007) Antimicrobial peptides, innate immunity, and the normally sterile urinary tract. J Am Soc Nephrol 18: 2810–2816. 5. McDermott AM (2009) The role of antimicrobial peptides at the ocular surface. Ophthalmic Res 41: 60–75. Imaging Bacterial Infection within Live Tissue Video S6 Two-photon imaging of cellular autofluorescence (magenta) and GFP bacteria (green) within an opaque infected wild-type mouse cornea. Bacteria are visible in both the epithelium and in the stroma, but the colonized areas are spatially separated by the intervening basal lamina. Those in the epithelium form a circular pattern around cells/cell nuclei, while those in the stroma are seen lined up (single file) along the collagen fibrils. (MOV) Video S7 Ex vivo blotted and bacteria-challenged MyD88 (2/2) mouse corneas at 8 h time-point. 3D image of the cornea showed GFP bacteria (green) traversed the entire thickness of the corneal epithelium (cytoplasmic autofluorescence, magenta). Traversal was accompanied by disruption of epithelial morphology, but bacteria did not cross the underlying intact basal lamina into the stroma. (MOV) Video S8 Ex vivo blotted and bacterially challenged MyD88 (2/2) mouse corneas at 4 h time-point. Traversal was not enabled at this shorter time-point, and the epithelium demonstrated normal morphology. However, the MyD88 (2/2) mouse corneas were found significantly more susceptible to bacterial adherence than wild- type at this early time-point. (MOV) Video S9 The reflection method was used to enable visualization of both live and dead cells (red), while the autofluorescence method showed only live cells (detects cytoplasmic NAD(P)H of actively metabolizing cells) (magenta). The two channels were superimposed (fuchsia) to confirm that penetrated bacteria (green) in tissue-paper blotted MyD88 (2/2) mouse corneas were actually overlaid with cells, and that some overlying cells remained viable. Note the large number of bacteria at the interface between the epithelium and the stroma (appears red) at the level of the basal lamina barrier. (MOV) Acknowledgments We thank Dr. Greg Barton (University of California, Berkeley) for C57BL/ 6 MyD88 (2/2) mice; Dr Gerald B. Pier (Harvard Medical School) for P. aeruginosa strain PAO1-GFP; H. Aaron (Molecular Imaging Center, UC Berkeley); and the Center for Host-Pathogen Studies, UC Berkeley for microscopy instrumentation and advice. Author Contributions Conceived and designed the experiments: CT DJE EAR SMJF. Performed the experiments: CT JL JJM PH. Analyzed the data: CT JL EAR DJE SMJF. Wrote the paper: CT JL EAR DJE SMJF. Developed the traversal quantification method: JL CT. 6. Fleiszig SM, Zaidi TS, Ramphal R, Pier GB (1994) Modulation of Pseudomonas aeruginosa adherence to the corneal surface by mucus. Infect Immun 62: 1799–1804. 7. Ramphal R, McNiece MT, Polack FM (1981) Adherence of Pseudomonas aeruginosa to the injured cornea: a step in the pathogenesis of corneal infections. Ann Ophthalmol 13: 421–425. 8. Meza I, Ibarra G, Sabanero M, Martinez-Palomo A, Cereijido M (1980) Occluding junctions and cytoskeletal components in a cultured transporting epithelium. J Cell Biol 87: 746–754. 9. Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, et al. (2010) Differential innate immune signalling via Ca(2+) sensor protein kinases. Nature 464: 418–422. 10. Gribar SC, Richardson WM, Sodhi CP, Hackam DJ (2008) No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation. Mol Med 14: 645–659. PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e24008