Fig 5. Visualization of bone’s 3-D lacunar canalicular system following non-aqueous optical clearing and mounting.

Results from the historical “gold-standard” technique of basic fuschin (BF) stained plastic embedded one-photon confocal microscopy are shown in the leftmost panel. Approximately 45-um of imaging penetration was achieved before canalicular detail was qualitatively lost in plastic embedded BF samples (shown by the red bar indicating “canalicular imaging penetration depth”). Results obtained following subsequent refinements in the en bloc imaging techniques that included optical clearing using MS or BABB, incorporation of two-photon microscopy, and the use of Villaneuva’s Osteochrome Bone Stain instead of BF, are shown in the panels to the right. The greatest improvements were achieved though the use of Osteochrome, BABB, and 2-P imaging. Similarly, with the “gold-standard” technique, visualization of lacunar details was restricted to ~50-um in depth due to optical “shadowing” within lacuna (compare the two leftmost panels). Optical clearing and 2-photon imaging also drastically improved the imaging of lacunar detail at depth.