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Polarization responses in Second Harmonic Generation (SHG) imaging microscopy are a valuable method to quantify aspects of tissue structure, and may be a means to differentiate normal and diseased tissues. Due to multiple scattering, the polarization data is lost in turbid tissues. Here we investigate if this information can be retained through the use of optical clearing which greatly reduces the scattering coefficient and increases the corresponding mean free path. To this end, we have measured the SHG intensity as a function of laser polarization and the SHG signal anisotropy in murine tendon and striated muscle over a depth range of 200 microns. We find that the laser polarization is highly randomized in the uncleared tissues at depths corresponding to only 2-3 scattering collisions (50- 10 microns). This depolarization of the laser is also reflected in the randomized anisotropy of the SHG signal as it is created over a range of polarization states. In strong contrast, both polarization signatures are significantly retained through ~200 microns of tissue thickness following treatment with 50% glycerol. Moreover, the measured polarization responses for both tendon and striated muscle are consistent with the extent of reduction of the respective scattering coefficients upon clearing. We suggest the method will be applicable to SHG imaging of connective disorders as well as cancer through several hundred microns of extracellular matrix.

Retention of polarization signatures in SHG microscopy of scattering tissues through optical clearing