Figure 1:

A) Schematic representation of the experimental set-up. As shown in the diagram, the excised metatarsal bone is maintained in place by a drop of cyanoacrylate glue at the proximal epiphysis on a a plastic spacer fixed at the bottom of a standard 3.5-cm plastic Petri dish. The shaft was held parallel to the bottom of the dish to allow for ISM scanning of the exposed bone cortex of the distal extremity. The dish was filled with the appropriate pre-warmed (37°C) medium (5.4 ml) and positioned on the viewing stage. The ISM was immersed in the medium after which the medium surface was immediately covered with a thin layer of light, clear, pre-warmed (37°C) mineral oil. The oil layer greatly reduces temperature-driven convection flow and helps maintain pH by minimizing air exchange at the surface of the solution. The ISM is then stabilized at reference (light blue ISM, away from the bone) and then moved close to the bone surface (dark blue ISM).