The program expects a colour image as input. The image will be displayed in the Image window. This window also contains the buttons for the overlay drawing tools on its left side. The routine info box provides guidance on what to do in each step, and allows setting the calibration of the pixel size.
The preliminary step is to identify the area to be analysed, normally the trabecular bone are, by drawing this using the polygon tool (highlighted in red).
Stage 1: Identifying the bone
The next step is the identification of the bone using colour thresholding using the sliders in the Threshold dialog box. The drawing tools can be used to edit the result.
The aniline blue stained bone is identified using colour thresholding. Cracks and holes are removed using binary opening and closings. Small particles are removed using an erode-reconstruct algorithm. Remaining defects can be edited manually
Stage 2: Identifying the osteoclasts
The osteoclasts are identified using the colour thresholding sliders in the “Resorption Surfaces” dialog, similar to the bone identification above. This box also allows to remove any TRAP-stained objects that are not close enough to the bone surface, and to merge particled that are close together, and therefore likely to be part of a single osteoclast. After completeion of this step the results are calculated and saved in an Excel compatible .csv file, as illustrated below: