Nonlinear Optical Microscopy Captures High-Resolution Images of Microstructures Within Three Types of Unlabeled Rat Cartilage

Cartilage is one of the most important tissues in mammalian growth and is classified into three types: hyaline, fibro, and elastic. To illustrate its potential as a clinical diagnostic tool, nonlinear optical microscopy (NLOM) was applied to simultaneously investigate images and spectra from three different types of rat cartilage. High-resolution images were obtained using the two-photon excited fluorescence and second-harmonic generation (SHG) of structures within fresh specimens. Since samples were imaged without hematoxylin and eosin (H&E) staining, detailed observations of tissue morphology and microstructural distribution were obtained without shrinkage or distortion. Nonlinear optical spectra confirmed that elastic cartilage is rich in elastic fibers, of which there are few in both hyaline and fibro cartilages. Structural dimensions were also measured with this technique, revealing the average area of the lacuna $(398.36 \pm 52.95\ \mu\text{m}^{2})$, or the average thickness of the territorial matrix $(0.83 \pm 0.26\ \mu\text{m})$, within elastic cartilage. Fourier analysis of SHG images was performed to further confirm that collagen in fibrocartilage showed significantly greater organization and directionality compared with collagen in hyaline cartilage $(p\ <\ 0.01)$. Our results show that each type of cartilage has different structural features and proportion of matrix, which may significantly contribute to pathology when damaged. Our findings demonstrate that NLOM holds the potential as a clinically relevant diagnostic tool for imaging degenerative tissues or assessing wound repair following cartilage injury.