Quantifying the backscattering of second harmonic generation in tissues with confocal multiphoton microscopy

The backward second harmonic generation (SHG) in mouse tissues is studied with a confocal multiphoton microscopy system. The total backward collected SHG (B-SHG) consists of the backward generated SHG and the backward-scattered forward-generated SHG (BS-SHG), which can be modeled by a Gaussian and a uniform distribution, respectively, at the confocal pinhole plane. By varying the pinhole size with a series of collection fibers, the proportion of the BS-SHG to the B-SHG and the proportion of BS-SHG to the forward generated SHG can be obtained. The approach is first validated by Monte Carlo simulation. It is then applied to two types of mouse tissues: mouse tail tendon and Achilles tendon. It is found that the BS-SHG contributes less to the B-SHG for the tail tendon than Achilles tendon with thicknesses of ∼300  μm . With the thickness of the Achilles tendon tissue increased to 1000 μm but the focal plane kept at the same depth, as high as ∼10% of the total forward SHG is backscattered and collected. The results indicate that BS-SHG may not be the major source of B-SHG in the tail tendon, but it may be the major source in the Achilles tendon. These methods and results provide a noninvasive method and supporting information for investigating the generation mechanism of SHG and help with optimizing backward SHG microscopy and spectroscopy measurements.