Presented at The Voice Foundation's 40th Annual Symposium: Care of the Professional Voice, Philadelphia, Pennsylvania, U.S.A., June 5, 2011.
Article first published online: 17 JAN 2012
Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.
Volume 122, Issue 2, pages 356–363, February 2012
How to Cite
Miri, A. K., Tripathy, U., Mongeau, L. and Wiseman, P. W. (2012), Nonlinear laser scanning microscopy of human vocal folds. The Laryngoscope, 122: 356–363. doi: 10.1002/lary.22460
This work was funded with a grant from the National Institutes of Health/NIDCD grant R01-DC005788, and the Natural Sciences and Engineering Research Council of Canada Discovery Grant. The authors have no other funding, financial relationships, or conflicts of interest to disclose.
- Issue published online: 23 JAN 2012
- Article first published online: 17 JAN 2012
- Manuscript Accepted: 24 OCT 2011
- Manuscript Revised: 7 OCT 2011
- Manuscript Received: 12 AUG 2011
- Human vocal folds;
- nonlinear laser scanning microscopy;
- laser microsurgery;
The purpose of this work was to apply nonlinear laser scanning microscopy (NLSM) for visualizing the morphology of extracellular matrix proteins within human vocal folds. This technique may potentially assist clinicians in making rapid diagnoses of vocal fold tissue disease or damage. Microstructural characterization based on NLSM provides valuable information for better understanding molecular mechanisms and tissue structure.
Experimental, ex vivo human vocal fold.
A custom-built multimodal nonlinear laser scanning microscope was used to scan fibrillar proteins in three 4% formaldehyde-fixed cadaveric samples. Collagen and elastin, key extracellular matrix proteins in the vocal fold lamina propria, were imaged by two nonlinear microscopy modalities: second harmonic generation (SHG) and two-photon fluorescence (TPF), respectively. An experimental protocol was introduced to characterize the geometrical properties of the imaged fibrous proteins.
NLSM revealed the biomorphology of the human vocal fold fibrous proteins. No photobleaching was observed for the incident laser power of ∼60 mW before the excitation objective. Types I and III fibrillar collagen were imaged without label in the tissue by intrinsic SHG. Imaging while rotating the incident laser light-polarization direction confirmed a helical shape for the collagen fibers. The amplitude, periodicity, and overall orientation were then computed for the helically distributed collagen network. The elastin network was simultaneously imaged via TPF and found to have a basket-like structure. In some regions, particularly close to the epithelium, colocalization of both extracellular matrix components were observed.
A benchmark study is presented for quantitative real-time, ex vivo, NLSM imaging of the extracellular macromolecules in human vocal fold lamina propria. The results are promising for clinical applications.