Figure 1
Lacuno-canalicular network and principles of THG-SHG-3PEF microscopy. (a) Schematic view of bone structure at the micrometric scale. Left, the network of interconnected osteocytes is embedded in the bone matrix and connected to Haversian channels containing nerves and blood vessels. Right, once the cells are removed, their previous location forms a network of porosities consisting of lacunae and canaliculi, forming the LCN. (b) Scheme of the microscope used for nonlinear imaging. An optical parametric oscillator (OPO) pumped by a titanium-sapphire laser (Ti:S) is focused and scanned inside the sample using galvanometric mirrors (XY) and a high-NA objective. Third harmonic generation (THG), second harmonic generation (SHG) and three-photon excited fluorescence (3PEF) signals are excited simultaneously and detected on photomultiplier tubes after spectral separation. 3PEF and SHG are epidetected while THG is collected in transmission through the sample. (c) Spectral representation (top) of the signals created by OPO excitation at λ = 1180 nm. THG (resp. SHG) is generated at a third (resp. half) of the excitation wavelength, while 3PEF is emitted in a broad range of wavelengths longer than λ/3. Bottom, corresponding simplified Jablonski diagrams. Dashed lines, virtual states. (d) THG signal for various sample geometries relevant for bone imaging. Interfaces (top) and heterogeneities down to a fraction of the size of the focal volume give rise to a localized signal, while a uniform medium produces no signal. Smaller size, randomly distributed heterogeneities create a non-zero, fluctuating signal.
