VITAL CONFOCAL MICROSCOPY IN BONE

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the me dial proximal tibial plateau of the rabbit, and special small, self-fo cussing objectives (dry 10/0.25, water immersion 20/0.45, and oil imme rsion 45/0.65 and 120/1.0) which mated and matched to the conical wind ow entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tis sue using two genera of confocal microscope: multiple aperture disc, t andem scanning, microscopes for observation in reflection, and video r ate confocal laser scanning microscopes for recording, mainly in the f luorescence mode. The latter allowed the study of a variety of intrave nously administered substances, including fluorescein, fluorescein-dex trans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluoresc ent markers, and replace them into the circulation. Calcein and tetrac ycline bind to the mineral front in bone: this labelling was studied i n progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation o f the system used both confocal fluorescence and scanning electron mic roscopy methods in the study of retrieved implants. These studies show ed that the subimplant cortical bone remodelled to a less compact stru cture with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, w ith the matrix containing large numbers of large cells: some of this t issue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide furthe r confirmation of the general utility of high-scan speed confocal meth odology in physiology.