Second Harmonic Imaging of Nasal, Auricular, and Costal Cartilage

There is little knowledge about the histological organization of facial and costal cartilages in terms of matrix structure and cell morphology. Second harmonic generation (SHG) imaging is a nonlinear imaging technique that capitalizes on signal generation from highly ordered macromolecules such as collagen fibers. The purpose of this study was to use SHG microscopy to image collagen extracellular matrix (ECM) structure, chondrocyte size, and density of these cartilages.

Experimental.

Surgical remnants of septal, lower lateral, rib, and auricular cartilages were collected following surgery, sectioned into 0.5-1 mm thick samples and fixed to facilitate batch process imaging. A Leica TCS SP8 MP Microscope and multiphoton laser were used to image the specimens. Images were analyzed for cell size, cell density, and collagen fiber directionality patterns using ImageJ.

SHG images of septal specimens show mesh-like structure of the ECM. There appears to be a superficial layer, characterized by flattened lacunae and middle zone, marked by circular lacunae clusters, similar to what is observed in articular cartilage. The structure of the ECM depicts a visible orientation perpendicular to the surface of the perichondrium. Cell size and density analysis through ImageJ suggests variety across cartilage types. Directionality analysis indicates that the collagen in the ECM displays preferred direction.

This study establishes clear extracellular models of facial and costal cartilages. Limitations include heterogeneous cartilage thickness due to processing difficulties. Further studies include automating the cutting process to increase uniformity of tissue thickness and increasing sample size to further validate results.

2 Laryngoscope, 133:3370-3377, 2023.