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Collagen Types I and II share highly conserved triple‐helical backbones and similar C-H stretch vibrational spectra, which limits the ability of conventional spectroscopic or second‐harmonic generation methods to unambiguously distinguish between them in native and engineered matrices. By combining polarization‐resolved sum‐frequency generation (SFG) measurements with tensor‐based simulations of the C-H stretch response, this work identifies collagen's asymmetric mode measured via the XXY tensor element as a robust optical marker that exhibits distinct spatial symmetries for collagen Type I and Type II. In rat auricular cartilage, analysis of the polarization‐resolved SFG signatures combined with vertex component analysis reveals pocket‐like domains of differently oriented collagen Type II fibrils rather than a uniformly aligned network. These findings establish polarization‐resolved SFG microscopy as a structurally specific tool for mapping collagen Type II architecture and label‐free discrimination of collagen Types I and II. 

Nonlinear optical (NLO) imaging platforms traditionally rely on refractive microscope objectives, which suffer from chromatic aberrations and temporal dispersion of pulsed excitation light. These issues degrade spatial imaging properties and signal brightness. Furthermore, the limited transmission range of refractive materials restricts NLO imaging, especially for applications requiring short- to mid-wave infrared excitation. While reflective microscope objectives offer an achromatic solution and broader transmission range (from visible to mid-infrared), conventional Schwarzschild designs have a central obscuration, which limits transmission throughput, imparts diffraction effects into the images, and, more generally, hinders the adoption of reflective optics in NLO microscopy. We overcome these obscuration-based limitations by developing a reflective microscope objective using freeform mirrors in a non-coaxial geometry. This obscuration-free design boasts a 0.65 numerical aperture (NA), near diffraction-limited imaging performance, and offers significantly improved transmission with wider fields-of-view. We demonstrate its utility by integrating it into a standard laser-scanning microscope and performing NLO microscopy across a wide range of excitation wavelengths. Our freeform microscope objective outperforms standard reflective designs, providing an achromatic, dispersion-free alternative to refractive lenses for NLO imaging.