Once the intestine was virtually unfolded, we could navigate through its volume, layer by layer (Fig. 6). Going inward from the outer boundary, the first layers of the intestine are characterized by the presence of the superficial vessels and the muscular tissue. Going deeper, a transition region, rich in muscular tissue and villi, is observed. Finally, the villi that protrude from the epithelial lining of the intestinal wall are reached and cut perpendicularly to their axis (Fig. 6, lower panel). In each 2-D unfolded image, hundreds of villi are sectioned, producing a significant assay of their morphology, density, and shape. The sampling of each layer is performed at a constant distance along the perimeter of the transverse sections of the specimen. This is crucial to reduce the deformations that would have been caused by projecting the sample to a simple cylindrical geometry. Indeed, the majority of the villi are cut transversally by the layers (Fig. 4), providing an accurate reconstruction of their shape in the new coordinate system. However, because of the irregular morphology of the tissue, some villi are cut obliquely or unevenly by the layer, particularly in the highly curved region of the sample. This can cause deformation of their shape (e.g., they can appear elliptical), but overall their number and density can still be measured accurately. The quantification of the villi number and density was accomplished in the new coordinate system using standard watershed segmentation (see Sec. 2). In Fig. 7, each segmented villus is shown in a different color. The separation between adjacent villi and the sharpness of the image indicates that not only the density and distribution of the villi can be obtained, but also their position and shape can be statistically analyzed. Looking at more details in the image of a layer and at the segmentation of the villi (colored panels in Fig. 7), we observe that the image quality varies along the horizontal direction. In fact, because of the unfolding, the center of the image corresponds to the portion of the sample closer to the microscope objective, while the extremities correspond to the back side. Hence, it is not surprising that the center of the image shows a resolution higher than the left and right borders: this difference is due to the scattering of the fluorescence emission while propagating through the sample toward the objective. Here, multiview acquisition would have improved the image quality of the dorsal region. However, the resolution obtained with a fast single-view scan was sufficient for accurate segmentation of the villi in the entire image.