Bone structure, THG microscopy setup and principle.

(a) simplified view of the osteocyte network in cortical bone (left) and the corresponding cavities in the bone matrix forming the lacuno-canalicular network (LCN). (b) Microscopy setup: a pulsed infrared 1180 nm beam is scanned and focussed on the sample, and the THG signal is collected in transmission onto a photomultiplier tube (PMT) using a 1.4NA condenser. (c) simplified Jablonski diagrams for single-photon excited fluorescence used in our confocal microscopy experiments (1PEF, left), and third-harmonic generation (THG, right). The THG signal is created at exactly one third of the excitation wavelength. Dotted line, virtual energy level. (d) the coherent buildup of the THG signal and the excitation phase shift of the excitation beam at the focal spot result in a non-zero signal only in the presence of optical heterogeneities within the focal volume, as created by canaliculi in the mineralized collagen bone matrix. This signal is strongly dependent on the canaliculi diameter and orientation.