Quantitative evaluation of the human vocal fold extracellular matrix using multiphoton microscopy and optical coherence tomography

Abstract

Identifying distinct normal extracellular matrix (ECM) features from pathology is of the upmost clinical importance for laryngeal diagnostics and therapy. Despite remarkable histological contributions, our understanding of the vocal fold (VF) physiology remains murky. The emerging field of non-invasive 3D optical imaging may be well-suited to unravel the complexity of the VF microanatomy. This study focused on characterizing the entire VF ECM in length and depth with optical imaging. A quantitative morphometric evaluation of the human vocal fold lamina propria using two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and optical coherence tomography (OCT) was investigated. Fibrillar morphological features, such as fiber diameter, orientation, anisotropy, waviness and second-order statistics features were evaluated and compared according to their spatial distribution. The evidence acquired in this study suggests that the VF ECM is not a strict discrete three-layer structure as traditionally described but instead a continuous assembly of different fibrillar arrangement anchored by predominant collagen transitions zones. We demonstrated that the ECM composition is distinct and markedly thinned in the anterior one-third of itself, which may play a role in the development of some laryngeal diseases. We further examined and extracted the relationship between OCT and multiphoton imaging, promoting correspondences that could lead to accurate 3D mapping of the VF architecture in real-time during phonosurgeries. As miniaturization of optical probes is consistently improving, a clinical translation of OCT imaging and multiphoton imaging, with valuable qualitative and quantitative features, may have significant implications for treating voice disorders.

Evaluation of the Human Vocal Fold Lamina Propria Development Using Optical Coherence Tomography

“…However, their position and dimensions closely match reported geometric data obtained for histology LP subdivisions (SLP, ILP, DLP) 43 . Furthermore, we previously demonstrated the ability to differentiate the VF subepithelial structures ex vivo with OCT and validated our findings with anatomical measurements made on fixed laryngeal tissue 58,59 . Moreover, numerous studies have already established the correlation of OCT imaging with light microscopy 35,60,61 …”

“…43 Furthermore, we previously demonstrated the ability to differentiate the VF subepithelial structures ex vivo with OCT and validated our findings with anatomical measurements made on fixed laryngeal tissue. 58,59 Moreover, numerous studies have already established the correlation of OCT imaging with light microscopy. 35,60,61 Our inability to provide a molecular landscape of the subepithelial VF, such as the extracellular matrix's fibrous protein composition, is another limitation.…”

Optical Microscopy and the Extracellular Matrix Structure: A Review

“…TPEF continues to be a powerful tool in the quantification of the ECM and has found several recent uses in mapping the ECM. In 2021, Benbouja and Hartnick used TPEF to quantify the morphology of the ECM in human vocal cords [ 77 ]. TPEF was used alongside other nonlinear optical methods to characterize the structure and organization of the ECM of the vocal folds, especially in targeting the elastic fibers found in the ECM.…”

“…TPEF was used alongside other nonlinear optical methods to characterize the structure and organization of the ECM of the vocal folds, especially in targeting the elastic fibers found in the ECM. They found that the ECM of human vocal folds has several distinct regions, which may explain why and where some pathologies occur and demonstrated the use of TPEF in mapping the architecture of the vocal fold in real-time during surgeries [ 77 ]. Hsaio et al used TPEF in their improved evaluation of liver fibrosis [ 71 ].…”

Characterization and Classification of Fibrillar Collagen Networks using Gray-Level Texture Analysis

“…These techniques have found extensive use in imaging the ECM, due to their suitability in visualizing structural components, such as fibrillar collagen, elastin, and fibronectin [57][58][59]. TPEF has found use in visualizing the structure of the ECM in human vocal cords, specifically the elastin fibers [60], and in imaging the structure of fibrotic liver tissue [61], making the technique useful in the characterisation of the ECM. While TPEF can still rely on exogenous fluorophores, it can also harness endogenous fluorophores, making it particularly suitable for live tissue imaging.…”