Figure 10: Work flow of image capture and the subsequent detection of the fluorescent signals. (A-D) represent microscope steps, while (E-H) are image processing steps. (A) Scan 1 captures mineralized bone and the fluorescent mineralization lines. (B) Scan 2 records the distribution of TRAP activity but loses the mineralization lines. (C) Scan 3 is performed after AP and DAPI staining. (D) Traditional hematoxylin stain for morphologic considerations, but this section is not part of the analysis. (E) Alignment of all the fluorescent images files into a single image stack and selection of the region of interest in the context of the whole bone. This step corrects for minor variation is rotation and shrinkage of the tape. (F) Enlarged view of the ROI prior to computer rendering. (G) Computer representation of the fluorescent features that are captured by the thresholding algorithms. (H) Projection of the features to the surface of the bone. This image is the basis of all the calculated measurements that are related to the bone surface. (I) and (J) are magnification of the boxed area of G and H. The enlarged figure demonstrates two trabeculae (grey signal) with bone formation (maroon colored AP overlying a red and green label) on the right side and resorption (yellow TRAP over non-mineralizing surface) on the opposite side.