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Peyer's patches (PPs) are typical gut-associated lymphoid tissues that are located along the wall of the small intestine and that serve as the major sites for generation of immunity to intestinal antigens. Their unique micro-organization is crucial for the generation of the immune response. Although many studies have been reported on the functional anatomy of PP, most investigations have relied on the random sampling of these organs, a procedure that is insufficient for the systemic scanning of the whole tissue or organ. By combining a variety of methods, we have accomplished 3D reconstructions of Peyer's patch. The complex reconstruction procedure includes several steps. First, the PP are serially sectioned at a thickness of 10 μm with a cryostat; (b) the serial sections are stained with haematoxylin–eosin; (c) multiple images from the PP are acquired with an automatic microscope and stitched together with Image Pro Plus to generate a composite image for the whole organ; (d) the serial images are reconstructed with Image J, Reconstruct and 3D Studio Max. The combinational approaches that we present here should be of value when extrapolated to the reconstruction of other tissues or organs. Moreover, the 3D model that we have created and our stereological analysis should be extremely helpful for further in vivo microscopic studies of PP with respect to the immune response.

The first step towards the three-dimensional (3D) reconstruction of histological structures from serial sectioned tissue blocks is the proper alignment of microscope image sequences. We have accomplished an automatic rigid registration program, named Image-Reg, to align serial sections from mouse lymph node and Peyer's patch. Our approach is based on the calculation of the pixel-correlation of objects in adjacent images. The registration process is mainly divided into two steps. Once the foreground images have been segmented from the original images, the first step (primary alignment) is performed on the binary images of segmented objects; this process includes rotation by using the moments and translation through the X, Y axes by using the centroid. In the second step, the matching error of two binary images is calculated and the registration results are refined through multi-scale iterations. In order to test the registration performance, Image-Reg has been applied to an image and its transformed (rotated) version and subsequently to an image sequence of three serial sections of mouse lymph node. In addition, to compare our algorithm with other registration methods, three other approaches, viz. manual registration with Reconstruct, semi-automatic landmark registration with Image-Pro Plus and the automatic phase-correlation method with Image-Pro Plus, have also been applied to these three sections. The performance of our program has been also tested on other two-image data sets. These include: (a) two light microscopic images acquired by the automatic microscope (stitched with other software); (b) two images fluorescent images acquired by confocal microscopy (tiled with other software). Our proposed approach provides a fast and accurate linear alignment of serial image sequences for the 3D reconstruction of tissues and organs.

The function of lymph nodes is greatly influenced by their unique microanatomy, in which distinct subpopulations of cells are compartmentalized by a meshwork of reticular cells and fibres, specialized blood and lymphatic vessels and nerves. Using antibodies against extracellular matrix (ECM) proteins (fibronectin, collagen IV and laminin), proteoglycan (perlecan), and a fibroblastic marker (ERTR-7), the distribution and molecular organization of the system of reticular fibres was investigated by three-dimensional (3D) reconstruction methods. Fibronectin, collagen IV and laminin are restricted to reticular fibres and have a similar distribution pattern, whereas perlecan is limited to the vascular system of the lymph node. Various compartments of the lymph node, such as the B-cell follicle, paracortex (including the high endothelial venules and paracortical cord), and medulla have been reconstructed to visualize their vasculature with respect to B and T cells. Since the morphology of lymph nodes may change significantly in pathological conditions, different compartments of reactive lymph node (after low-dose Listeria monocytogenes infection), especially germinal centres, were also investigated. The data presented here should facilitate our understanding of the 3D organization of non-immune cell components of lymph nodes, which is crucial for cell adhesion, migration, activation, and differentiation in normal and pathological conditions.

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane–membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.

Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.