Three-dimensional (3D) reconstruction of the AV junction
All sections collected from 80 groups (320 sections) from one rat were used to reconstruct a 3D computational model of the anatomy of the AVN. 3D-DOCTOR (version 4.0; Able Software Corp.) and Adobe Photoshop were used to analyze the images and construct a 3D anatomical model of the AVN. There were four steps in this process. Step 1: Segmentation. Various tissue types could be identified in Masson’s trichrome-stained sections: myocytes were stained purple, and connective tissues were stained blue. Further cell types were identified by merging adjacent immunofluorescence-labeled images with images from similarly grouped Masson’s trichrome-stained sections using Adobe Photoshop, and these combinations included the following: (i) Cx43-negative, Cx40-negative, HCN4-positive and strongly Cx45-positive nodal tissues; (ii) Cx43-negative, Cx40-positive, HCN4-positive and strongly Cx45-positive His bundle tissues; and (iii) Cx43-positive ventricular and atrial muscles. Then, Adobe Photoshop was used to outline various cell types (carried out manually) on images obtained from Masson’s trichrome-stained sections. A unique color was used to highlight each cell type. Finally, multicolor images were converted into grayscale images in preparation for 3D reconstruction. The result was a two-dimensional model of sections showing the distribution of myocytes, connective tissues and different cell types. Step 2: Alignment.Sections were aligned by some combination of translation, rotation and scaling, but the shape of the sections was left unchanged. The alignment of sections is crucial in developing a model. Tracts of the nodal tissue, tricuspid valve and bottom edge of the preparation were the main landmarks used to align the sections. Each image was aligned by 3D-DOCTOR and saved. The saved aligned images were used in preparation for the next step of 3D reconstruction. Step 3: Calibration . The voxel size (image resolution) must be provided to obtain a 3D image so that the 3D rendering has the correct scale in all three dimensions, enabling it to incorporate the reported measurements. For the voxel resolution, the X and Y values are equivalent to the size of one pixel in a slice, while the Z value is the slice thickness and distance between the centers of two neighboring slices in 3D space. A group at the same level of the tissues was created as an assumption; thus, the distance between two prepared grayscale images was 24/28 μm (6/7 μm*4). Volume correlation measurements were based on parameters obtained from the image and scale plate provided by the camera (1 μm = 1.17 unit). As a result, Z values (28.08/32.76 units) were used as the thickness between the two groups of sections, which is an option for 3D reconstruction. Step 4: Model visualization. After creating an interactive segment, surface rendering was performed using 3D-DOCTOR. Although only two preparations were used to generate an anatomical model, we confirmed these anatomical findings in four other preparations.