Abstract
A confocal multiphoton microscopy system with various detection pinholes was used to differentiate backward scattered second harmonic generation (BS-SHG) from backward generated SHG (BG-SHG) based on the fact that BS-SHG is more scattered and therefore has a much bigger spot size than BG-SHG. BS-SHG is quantified from two types of mouse tissues, such as Achilles tendon, and skin, and at various focal depths. It is found that the BS-SHG contributes less to the total backward SHG for the skin than Achilles tendon with thicknesses of around three hundred micrometers. For tissue with larger F/B intensity ratio such as Achilles tendon, increasing the tissue thickness reduces it tremendously. However, for tissue with smaller F/B intensity ratio, tissue thickness increment does not alter it significantly. In addition, larger F/B intensity ratio might be related with a greater scattering coefficient from our Achilles tendon and skin comparison. When the focal point is moved deeper into tissue, the contribution of BS-SHG is found to decrease due to a reduced pass length of the forward propagated photons. On the contrary, when the tissue thickness increases, the contribution of the BS-SHG is increased. These observations for thicker skin tissues are related with our F/B intensity ratio measurement for thin mouse skin sample in terms of that the magnitude of backward generated SHG are dominant among the total backward SHG in mouse skin tissue. Considering the phase mismatching condition in the forward and backward directions, these results may indicate that quasi-phase matching originating from the regular structure of collagen could help with reducing the phase mismatch especially in the backward direction.
