Second harmonic generation (SHG) imaging microscopy was used to investigate the collagen matrix organization within bone tissue. A SHG and two-photon excitation fluorescence (2PEF) microscope was used for this study. The SHG/2PEF microscope was based on a Leica TCS STED CW, Resonant Scanner Spectral confocal scanning head (Leica Microsystems, Mannheim, Germany). The SHG is collected both in back-scattering (BSHG) and in a transmitted light configuration (forward second harmonic generation; FSHG). Since the excitation wavelength is set at 860 nm, the BSHG spectral component of interest, 415–445 nm, is selected by means of the Leica built-in prism based spectrophotometer, whereas the FSHG is selected by two filters, an IR blocking filter and a 430/30 nm band-pass color filter (HQ430/30 m, Chroma Technology Corp., Bellows Falls, VT, USA). All panels have been acquired using a 76 nm pixel size and a 100x 1.4 NA oil-immersion objective (HCX PL APO 100x/1.4 Oil, Leica Microsystems, Mannheim, Germany). SHG imaging was used to gather specific organizational motifs, such as the structure of endogenous collagen proteins26. Moreover, due to the coherent nature of SHG signal, the intensity of the output is proportional to the square of the local concentration of the proteins observed and is intrinsically 3D.