Detection of fluorescently labeled <i>V. cholerae</i> in the intact infant mouse small intestine by intravital two-photon microscopy.


<p>(a) Schematic representation (left) of the surgical intravital imaging approach to visualize intestinal tissue by two-photo microscopy in live anesthetized mice following orogastric inoculation with <i>V. cholerae</i>. Transverse view of exteriorized intestinal loop (right), depicting objective focal path from the serosal side through the various tissue layers towards the intestinal villus. The resulting focal plane is a tangential section along the intestine as depicted in (b–d). (b) Sequential two-photon micrographs (XY plane) taken from a 3-dimensional Z-stack of the SI, revealing penetration depths for two-photon excitation of up to 150 µm below the serosa layer. Depths (Z axis) 0 to 50 µm below the serosa include crypt regions, and >100 µm encompass the villi region. Blue denotes collagen fibers, Magenta denotes blood vessels. Scale bar 50 µm. (c) Differential localization of <i>V. cholerae</i> within the crypt or villus regions in the proximal, medial, and distal segments of the SI. Top row panel displays representative intravital two-photon micrographs of the crypt region for the proximal SI segment, and of the villus region for the medial and distal SI segments, of non-infected mice. Bottom row panel displays representative intravital two-photon micrographs of the crypt region and villus regions for the proximal, medial, and distal regions of the SI following orogastric inoculation with VcGreen and VcRed, respectively. Blue denotes collagen fibers, Green denotes colonies of VcGreen, Red denotes colonies of VcRed. Scale bar 10 µm. (d) Visualization of VcGreen in explanted distal SI segments. Representative two-photon micrographs taken from time-lapsed movies (see Supplemental <a href="" target="_blank">Video S1</a>, <a href="" target="_blank">Video S2</a>) show single VcGreen cells in the intravillus space and discrete microcolonies of VcGreen associated with the villus epithelium. White/Yellow denotes autofluorescence, Green denotes VcGreen. Scale bar 10 µm.</p

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