Method Development: Characterization of the Structure of the Thymic Epithelial Cell Network Utilizing Fluorescent Whole Slide Scanning and 3D SEM Array Tomography

The thymus is a primary lymphoid organ for T cell maturation. It consists of an outer (cortex) and an inner (medulla) zone, which regulate the maturation of functional T cells and establishment of central tolerance, respectively. Within the thymus, developing T cells (a.k.a., thymocytes) commit to a sequence of developmental stages, orchestrated by an interlacing network of cortical and medullary thymic epithelial cells (cTEC and mTEC, respectively), which provide the essential nutrients, and niche factors to the developing thymocytes [1]. The thymus is the most sensitive organ to cytoreductive treatments, and it rapidly involutes after chemotherapy insult in pediatric cancer patients. As a consequence, pediatric cancer survivors develop hindered immunity, persistent/mortal infections and autoimmune disease. Because future regenerative strategies in chemotherapy-induced thymic involution will depend upon promoting the regeneration and health of cTEC/mTEC compartments, it is important to establish an imaging approach to assess the functional integrity of thymic epithelium in preclinical mouse models. In order to characterize the overall structure of the cTEC compartment, mouse thymus 5 um sections were stained to visualize nuclei (DAPI) and Keratin-8 (KRT8) and the whole tissue section was automatically scanned with a 3DHISTECH P250 Flash III automated slide scanner (Figure 1A). 40X individual fields of view of the cortical region were subsequently captured using CaseViewer v2.4 (3DHISTECH) and exported as TIFF images. Further processing occurred with ImageJ. Regions of interest (ROI) were designated, the KRT8+ signal was binarized using intensity thresholding, and the KRT8+ fraction was calculated. Furthermore, these images were despeckled, skeletonized (with no loop or end-point elimination) and then subjected to the “Analyze Skeleton” plugin in ImageJ [2] (Figure B-E). The output collected was the number of branches, number of junctions, both of which were expressed per μm2 of thymic cortex, as well as average branch length. To further investigate the structure of the interlacing network of cTEC/mTEC at higher resolution and in 3D, we decided to use serial section array tomography to visualize the three-dimensional structure of this network. Serial sectioning allows for visualization of the extent of the wrapping of the network around the T cells. Samples were prepared using the National Center for Microscopy and Imaging method of OTO (Osmium-Thiocarbohydrazide-Osmium) processing [3]. 55nm serial sections were cut using a Leica ARTOS microtome equipped with a Diatome AT-4 knife and were picked up on silicon wafers. Sections were examined in a Zeiss Supra Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy, LLC North America) in backscatter mode, using an accelerating voltage of 8 KV. Images were collected using Atlas (Zeiss) with an X/Y pixel size of 6nm and a 6µs dwell time. Data reconstruction and 3-D modeling was done using ETOMO and 3DMOD, applications bundled with the IMOD package [4]. Analysis procedure for quantification of branching capacity of the cTEC. A. Overview of scanned whole piece of thymus tissue. Region of interest shown in subsequent panels is outlined in white. B. Region of interest showing DAPI nuclei (cyan) and KRT8 (red). C. Region of interest showing single channel KRT8 (red) used for further analysis. D. Initial binary image constructed from contours. E. Further processed final binary image. Three-dimensional reconstruction of cTEC network, shown in blue and green, with multicolored nuclei of developing T cells shown surrounded by the network. A. Clipped model shown with backscatter image of thymus tissue. B. Full three-dimensional model with transparent nuclei of T cells.