Fig. 4

The PEGASOS method provides an approach for investigating hard tissue in 3-D. Hard tissue samples were harvested from adult mice and processed following the passive immersion procedure for hard tissue organs. Half of the skull was imaged with a stereomicroscope before (a) and after clearing (a’). b The cleared skull bone was imaged based on the SHG signal acquired with a 5× objective on a two-photon microscope. The intact mandible (c) and femur (e) were imaged. Optical sections were acquired at boxed regions and enlarged in c’ and e’ respectively. Boxed areas in c’ and e’ were re-imaged with a 32× 0.85 NA objective to reveal the trabecular bone organization of the alveolar bone (d) or femur (f). g Tooth within the mandible was imaged with a two-photon microscope. Optical section was obtained to show the pulp chamber (g’). h and h’ An intact knee joint was cleared. Optical sections acquired at different depth are displayed. Arrows indicate the articular surface. Articular ligaments are labeled. SHG second harmonic generation signal