Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory

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

Immunohistochemical study of the reticular and vascular network of mouse lymph node using vibratome sections

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

Automatic registration of serial sections of mouse lymph node by using Image-Reg

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

Serial sectioning and three-dimensional reconstruction of mouse Peyer's patch

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

Surface modification of closed plastic bags for adherent cell cultivation

In modern medicine human mesenchymal stem cells are becoming increasingly important. However, a successful cultivation of this type of cells is only possible under very specific conditions. Of great importance, for instance, are the absence of contaminants such as foreign microbiological organisms, i.e., sterility, and the chemical functionalization of the ground on which the cells are grown. As cultivation of these cells makes high demands, a new procedure for cell cultivation has been developed in which closed plastic bags are used. For adherent cell growth chemical functional groups have to be introduced on the inner surface of the plastic bag. This can be achieved by a new, atmospheric-pressure plasma-based method presented in this paper. The method which was developed jointly by the Fraunhofer IST and the Helmholtz HZI can be implemented in automated equipment as is also shown in this contribution. Plasma process gases used include helium or helium-based gas mixtures (He + N2 + H2) and vapors of suitable film-forming agents or precursors such as APTMS, DACH, and TMOS in helium. The effect of plasma treatment is investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements and XPS. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, respectively, reacting with surface radical centers formed during contact with the discharge. Primary amino groups are obtained on the inner surface by treatment in mixtures with nitrogen and hydrogen albeit their amount is comparably small due to diffusion of oxygen through the gas-permeable bag, interfering with the plasma-amination process. Surface modifications introducing amino groups on the inner surface turned out to be most efficient in the promotion of cell growth

Surface modification of closed plastic bags for adherent cell cultivation

In modern human mesenchymal stem cells are gaining increasing importance. Usein helium-based gas mixtures (He+N2+H2) as well as helium enriched with suitable film- forming agents the inner surface of polymer bags can be modified with a plasma process development at Fraunhofer IST. The effect of plasma treatment in investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, resp., reacting with surface radical centres formed in the discharge. For the same reason the area density of primary amino groups on the surface obtained by treatment in mixtures with nitrogen is comparably small. With regard to their applicability in cell cultivation three different coatings (bases on APTMS, DACH and TMOS) were tested. It is shown that primary amino groups play an important role in cell cultivation of adherent growing bone marrow mesenchymal stem cells

Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells

Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase-deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence-associated β-galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration