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

Fouzi Benboujja,Christopher Hartnick

doi:10.1038/s41598-021-82157-9

Fouzi Benboujja, Christopher Hartnick

Open Access

https://doi.org/10.1038/s41598-021-82157-9

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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.

Highlights

Identifying distinct normal extracellular matrix (ECM) features from pathology is of the upmost clinical importance for laryngeal diagnostics and therapy
Optical imaging can assess the vocal fold (VF) under various physiological and pathological conditions heretofore unexplained, which cannot be accomplished with traditional imaging techniques such as ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT)
This study focused on the optical contrast of VF ECM with an eye toward real-time phonosurgery guidance

Summary

Introduction

Identifying distinct normal extracellular matrix (ECM) features from pathology is of the upmost clinical importance for laryngeal diagnostics and therapy. 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. Microlaryngeal techniques have been developed to preserve, to a maximum extent, the microanatomy of the VF, and its composition has been extensively studied with histopathology a nalysis[1,2,6,7,8,9] and scanning electron microscopy (SEM)[10,11] Despite these efforts, it remains challenging to extend such two-dimensional histological investigations to the patient’s structure-functional anatomy. 3D imaging modalities such as ultrasound (US)[12], computed tomography (CT)[13,14], and magnetic resonance imaging (MRI)[15] have been investigated on the VF mucosa; their findings all report the insufficient spatial resolution to visualize micron-scale structures of the ECM

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