Preliminary evaluation of noninvasive microscopic imaging techniques for the study of vocal fold development

Understanding pediatric voice development and laryngeal pathology is predicated on a detailed knowledge of the microanatomy of the layered structure of the vocal fold. Our current knowledge of this microanatomy and its temporal evolution is limited by the lack of pediatric specimen availability. By providing the capability to image pediatric vocal folds in vivo, a noninvasive microscopy technique could greatly expand the existing database of pediatric laryngeal microanatomy and could furthermore make longitudinal studies possible. A variety of natural-contrast optical imaging technologies, including optical frequency domain imaging (OFDI), full-field optical coherence microscopy (FF-OCM), and spectrally encoded confocal microscopy (SECM) have been recently developed for noninvasive diagnosis in adult patients. In this paper, we demonstrate the potential of these three techniques for laryngeal investigation by obtaining images of excised porcine vocal fold samples. In our study, OFDI allowed visualization of the vocal fold architecture deep within the tissue, from the superficial mucosa to the vocalis muscle. The micron-level resolution of SECM allowed investigation of cells and extracellular matrix fibrils from the superficial mucosa to the intermediate layer of the lamina propria (LP) (350 microm penetration depth). The large field of view (up to 700 microm), penetration depth (up to 500 microm), and resolution (2x2x1microm [XxYxZ]) of FF-OCM enabled comprehensive three-dimensional evaluation of the layered structure of the LP. Our results suggest that these techniques provide important and complementary cellular and structural information, which may be useful for investigating pediatric vocal fold maturation in vivo.