Fluorescence microscopy imaging of bone for automated histomorphometry.

We have developed a computer-based method for the automated quantification of bone tissue in histological sections of decalcified specimens. Bone tissue was generated by ectopic implantation of ceramic-based carriers loaded with human bone marrow stromal cells (BMSCs). The method is based on the acquisition of multimodal images, in order to identify and measure the area covered by bone tissue (using fluorescent light) and the total area of tissue (using transmitted light), thereby excluding the regions corresponding to nonresorbed scaffold. The amount of bone as a percentage of the total area of interest (bone/area) and of the newly formed tissue (bone/tissue) is automatically derived. The computer-based results correlated closely with those obtained by manual identification of bone and tissue areas in the same histological fields (R2 = 0.997; p < 0.0005), with errors dependent on the magnification used but always lower than 9.4%. The method was used to compare the bone/tissue and bone/area percentages in...