Wavelength and polarization optimization through Stokes parameters for second harmonic generation microscopy in...

Second harmonic generation (SHG) microscopy has experienced a research expansion from fundamental collagen studies in tendon and skin to a growing body of work on ocular tissues, where the label-free and high-resolution aspects have offered unique possibilities. Nevertheless, polarization states and excitation wavelength have not been investigated for corneal samples. Here, we present a comprehensive investigation of SHG microscopy applied to fibrillar collagen from murine corneal tissue. Emphasis is given to polarization control, cross-polarization effects, and excitation wavelength optimization. Precise characterization of the polarization state at multiple locations along the optical path via Stokes parameter measurements revealed significant polarization degradation introduced by optical components not typically considered as polarizing. Polarization degeneration was compensated by rotating the polarization axis to a microscope-specific angle, restoring a near-ideal linear polarization state at the sample plane. Numerical simulations using Wavesim software demonstrate that cross-polarized field components scale as ∼NA⁴, highlighting their non-negligible contribution to SHG signals acquired at high numerical apertures. Spectroscopic SHG measurements of murine corneal tissue confirm that SHG is the dominant nonlinear signal source within the investigated excitation range, with the signal-to-noise ratio following a cubic dependence on excitation wavelength. Accounting for detector sensitivity, optical component transmission, and the intrinsic UV absorption of ocular tissue, 800 nm emerges as the most practical excitation wavelength for SHG imaging of the cornea. Our findings establish practical guidelines for optimizing excitation conditions and polarization management in SHG microscopy, advancing quantitative structural imaging of collagenous biological tissues.