Second Harmonic Imaging of Nasal, Auricular, and Costal Cartilage.

OBJECTIVE: 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. STUDY DESIGN: Experimental. METHODS: 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. RESULTS: 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. CONCLUSION: 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. LEVEL OF EVIDENCE: 2 Laryngoscope, 133:3370-3377, 2023.

Fast, large area multiphoton exoscope (FLAME) for macroscopic imaging with microscopic resolution of human skin.

We introduce a compact, fast large area multiphoton exoscope (FLAME) system with enhanced molecular contrast for macroscopic imaging of human skin with microscopic resolution. A versatile imaging platform, FLAME combines optical and mechanical scanning mechanisms with deep learning image restoration to produce depth-resolved images that encompass sub-mm2 to cm2 scale areas of tissue within minutes and provide means for a comprehensive analysis of live or resected thick human skin tissue. The FLAME imaging platform, which expands on a design recently introduced by our group, also features time-resolved single photon counting detection to uniquely allow fast discrimination and 3D virtual staining of melanin. We demonstrate its performance and utility by fast ex vivo and in vivo imaging of human skin. With the ability to provide rapid access to depth resolved images of skin over cm2 area and to generate 3D distribution maps of key sub-cellular skin components such as melanocytic dendrites and melanin, FLAME is ready to be translated into a clinical imaging tool for enhancing diagnosis accuracy, guiding therapy and understanding skin biology.