Collagen and myosin characterization by orientation field second harmonic microscopy

Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor χ(2) can be determined for each pixel of the image. The ratio ρ=χ33/χ15 of the principal components of χ(2) of collagen and myosin was obtained with the same method, and found within the range 1.6–1.8 and 0.5–0.6 respectively. The orientation of the principal axis of χ(2) is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).