High frequency ultrasonic characterization of human vocal fold tissue

Recently, endolaryngeal sonography at frequencies ranging from 10 to 30 MHz has been found to be useful in diagnosing diseases of the vocal folds (VFs). However, image resolution can be further improved by ultrasound at higher frequencies, necessitating the measurement of high-frequency acoustic properties of VF tissue. The ultrasonic parameters of integrated backscatter, sound velocity, and frequency-dependent attenuation coefficient were measured in both the lamina propria (LP) and vocalis muscle (VM) of human VFs using a 47 MHz high-frequency ultrasonic transducer. The integrated backscatter was -173.44+/-6.14 (mean+/-s.d.) and -195.13+/-3.58 dB in the LP and VM, respectively, the sound velocity was 1667.68+/-44.9 and 1595.07+/-39.33 ms, and the attenuation coefficient at 47 MHz was 8.28+/-1.72 and 7.17+/-1.30 dBmm. The difference between these ultrasonic parameters may be attributed to variations in the structure and fiber concentrations in VF tissue. These results could serve as a useful clinical reference for the further development of high-frequency ultrasound devices for endolarynx sonography applications.     

A constitutive model of the human vocal fold cover for fundamental frequency regulation

The elastic as well as time-dependent mechanical response of the vocal fold cover (epithelium and superficial layer of the lamina propria) under tension is one key variable in regulating the fundamental frequency. This study examines the hyperelastic and time-dependent tensile deformation behavior of a group of human vocal fold cover specimens (six male and five female). The primary goal is to formulate a constitutive model that could describe empirical trends in speaking fundamental frequency with reasonable confidence. The constitutive model for the tissue mechanical behavior consists of a hyperelastic equilibrium network in parallel with an inelastic, time-dependent network and is combined with the ideal string model for phonation. Results showed that hyperelastic and time-dependent parameters of the constitutive model can be related to observed age-related and gender-related differences in speaking fundamental frequency. The implications of these findings on fundamental frequency regulation are described. Limitations of the current constitutive model are discussed.     

The shear modulus of the human vocal fold in a transverse direction

The aim of this study was to measure the shear modulus of the vocal fold in a human hemilarynx, such that the data can be related to direction of applied stress and anatomical context. Dynamic spring rate data were collected using a modified linear skin rheometer using human hemilarynges, and converted to estimated shear modulus via application of a simple shear model. The measurement probe was attached to the epithelial layer of the vocal fold cover using suction. A sinusoidal force of 3g was applied to the epithelium, and the resultant displacement logged at a rate of 1kHz. Force measurement accuracy was 20microg and position measurement accuracy was 4microm. The force was applied in a transverse direction at the midmembranous point between the vocal process and the anterior commissure. The shear modulus of the three female vocal folds ranged from 814 to 1232Pa. The shear modulus of the three male vocal folds ranged from 1021 to 1796Pa. These data demonstrate that it is possible to obtain estimates for the shear modulus of the vocal fold while preserving anatomical context. The modulus values reported here are higher than those reported using parallel plate rheometry. This is to be expected as the tissue is attached to surrounding structures, and is under natural tension.     

Effects of long-term intubation on vocal fold mucosa in dogs

A canine model was used to study effects of long-term intubation on vocal fold mucosa. Dogs' larynges were removed 5 weeks after a 7-day intubation period and were compared with control tissue. Intubation effects on vocal fold mucosa were highly variable. Most severe damage was observed posteriorly, at the presumed location of direct tube-mucosa contact. Effects judged to be less severe but still significant were noted in tissue anterior to this site. Morphometric analysis of the layers of the intubated mucosa revealed significant differences in epithelium, connective tissue, and glands, as compared with control tissue. Differences were also observed for blood vessels and nerves. Of particular clinical importance was evidence of damage along membranous, as well as cartilaginous, portions of the true vocal fold, and of damaged connective tissue and cartilage underlying epithelium which appeared normal. Implications of the findings for recovery from intubation, and for voice, are discussed.     

Effects of ambient inhaled ozone on vocal fold mucosa in bonnet monkeys

Deleterious effects of ambient ozone (O₃) on the nasal and lower airways is well documented. However, no investigations have considered possible effects on vocal fold mucosa. To this end, six macaque monkeys were exposed to 0.64 ppm O₃ for 8 h/day for a total of 7 days. Three monkeys were killed 12 h postexposure, and the other three at 7 days postexposure. Larynges were excised and subjected to histological staining and morphometric analyses. Results revealed increases in the thickness of epithelial tissue in the 12 h group. In the 7 day group, differences in both epithelial and lamina propria thicknesses were noted. Additional changes consistent with an initial inflammatory response, with associated disruption in glands and vessels, followed by some evidence of healing, were also noted. Implications of the data for the larynx and voice quality in humans exposed to similar and other environmental toxins are discussed.     

Oral breathing increases Pth and vocal effort by superficial drying of vocal fold mucosa.

Oral breathing superficially dehydrates the airway lumen by decreasing the depth of the sol layer in humans and animals. Conversely, nasal breathing can increase the humidity of inspired air. We compared the effects of short-term oral and nasal breathing on Pth and perceived vocal effort in 20 female subjects randomly assigned to two groups: oral breathing (N = 10, age 21-32 years); nasal breathing (N = 10, age 20-36 years). We hypothesized that short-term oral breathing, but not nasal breathing, would increase Pth, and that subjects would perceive this change as an increase in vocal effort. Following 15 minutes of oral breathing, Pth increased at comfortable and low pitch (p < 0.01) with 6 of 10 subjects reporting increased vocal effort. Nasal breathing reduced Pth at all three pitches (p < 0.01), and 7 of 10 subjects reported decreased vocal effort. Over all subjects, 49% of the variance in treatment-induced change in Pth was accounted for by change in vocal effort (R = 0.70). We posit that obligatory oral breathing places healthy subjects at risk for symptoms of increased vocal effort. The facilitatory role of superficial hydration on vocal fold oscillation should be considered in biomechanical models of phonation and in the clinical prevention of laryngeal dryness.     

Preliminary report on hormone receptors in the human vocal fold

There has been an ongoing effort to describe the physiologic factors associated with perceived and/or measured human voice changes that occur with age. In our study we focused on possible endocrine involvement on voice by using immunohistochemical staining to observe hormone receptor presence in vocal folds from 42 deceased subjects (fresh cadavers), male and female, ranging in age from 2 months to 82 years (average 37.7 years). On statistical analysis, age and gender were found to be associated with progesterone receptor staining of the glandular nuclei (young>old P = 0.013; male>female, P = 0.060). Gender was associated with androgen receptor staining in glandular cytoplasm (male>female, P = 0.014) and progesterone receptor staining in the epithelial cytoplasm (male>female, P = 0.039). No statistical significance was noted in other categories. CONCLUSION: Hormone receptors are found in the nucleus and cytoplasm of cells in the vocal fold with statistically significant differences in age and gender distribution.     

Investigating acoustic correlates of human vocal fold vibratory phase asymmetry through modeling and laryngeal high-speed videoendoscopy

Vocal fold vibratory asymmetry is often associated with inefficient sound production through its impact on source spectral tilt. This association is investigated in both a computational voice production model and a group of 47 human subjects. The model provides indirect control over the degree of left-right phase asymmetry within a nonlinear source-filter framework, and high-speed videoendoscopy provides in vivo measures of vocal fold vibratory asymmetry. Source spectral tilt measures are estimated from the inverse-filtered spectrum of the simulated and recorded radiated acoustic pressure. As expected, model simulations indicate that increasing left-right phase asymmetry induces steeper spectral tilt. Subject data, however, reveal that none of the vibratory asymmetry measures correlates with spectral tilt measures. Probing further into physiological correlates of spectral tilt that might be affected by asymmetry, the glottal area waveform is parameterized to obtain measures of the open phase (open/plateau quotient) and closing phase (speed/closing quotient). Subjects' left-right phase asymmetry exhibits low, but statistically significant, correlations with speed quotient (r=0.45) and closing quotient (r=-0.39). Results call for future studies into the effect of asymmetric vocal fold vibration on glottal airflow and the associated impact on voice source spectral properties and vocal efficiency.     

Investigation of vocal fold impact stress in human subjects

The primary purpose of the study was to explore a methodology for measuring vocal fold impact stress (SI) in awake humans, and to provide information about the general magnitude of SIs that may occur at the midpoint of the membranous vocal folds during phonation. A secondary purpose was to examine the potential use of the electroglottographic closed quotient (EGG CQ) to indirectly reflect SI. Seven male and 13 female adults were enrolled as subjects, of whom 18 had normal larynges and normal voices, 1 had nodules, and 1 had vocal fold paresis and bowing. Subjects attempted to produce 3 different voice types (pressed, normal, breathy), at 3 different pitches (low, medium, high) and 3 different loudness levels (quiet, medium, loud). For a first set of trials, only EGG data were collected. For a second set, a sensor was also introduced to the midmembranous glottis for the collection of SI data. The primary findings were that (1) endolaryngeal sensor placement was achieved during phonation trials for 17 of 20 subjects; however, grossly consistent anteroposterior positioning was accomplished, and analyzable data were obtained, for only 7 subjects; (2) SIs ranged from less than 1 kPa to about 3 kPa for those 7 subjects; and (3) no relation was detected between simultaneous CQs and SIs for individual data, although a relation was reported in a prior canine study. One possible reason for the failure to show such a relation in the present study was subtle variations in vertical as well as anteroposterior positioning of the sensor during the trials. Future studies should focus on developing a methodology for ensuring invariant 3-dimensional sensor positioning between the membranous folds, so that the stability of both SI and simultaneous CQ data can be improved.     

Simulation based estimation of dynamic mechanical properties for viscoelastic materials used for vocal fold models

In order to obtain a deeper understanding of the human phonation process and the mechanisms generating sound, realistic setups are built up containing artificial vocal folds. Usually, these vocal folds consist of viscoelastic materials (e.g., polyurethane mixtures). Reliable simulation based studies on the setups require the mechanical properties of the utilized viscoelastic materials. The aim of this work is the identification of mechanical material parameters (Young's modulus, Poisson's ratio, and loss factor) for those materials. Therefore, we suggest a low-cost measurement setup, the so-called vibration transmission analyzer (VTA) enabling to analyze the transfer behavior of viscoelastic materials for propagating mechanical waves. With the aid of a mathematical Inverse Method, the material parameters are adjusted in a convenient way so that the simulation results coincide with the measurement results for the transfer behavior. Contrary to other works, we determine frequency dependent functions for the mechanical properties characterizing the viscoelastic material in the frequency range of human speech (100–250 Hz). The results for three different materials clearly show that the Poisson's ratio is close to 0.5 and that the Young's modulus increases with higher frequencies. For a frequency of 400 Hz, the Young's modulus of the investigated viscoelastic materials is approximately 80% higher than for the static case (0 Hz). We verify the identified mechanical properties with experiments on fabricated vocal fold models. Thereby, only small deviations between measurements and simulations occur.     

Phonation threshold pressure: comparison of calculations and measurements taken with physical models of the vocal fold mucosa

In an important paper on the physics of small amplitude oscillations, Titze showed that the essence of the vertical phase difference, which allows energy to be transferred from the flowing air to the motion of the vocal folds, could be captured in a surface wave model, and he derived a formula for the phonation threshold pressure with an explicit dependence on the geometrical and biomechanical properties of the vocal folds. The formula inspired a series of experiments [e.g., R. Chan and I. Titze, J. Acoust. Soc. Am 119, 2351-2362 (2006)]. Although the experiments support many aspects of Titze's formula, including a linear dependence on the glottal half-width, the behavior of the experiments at the smallest values of this parameter is not consistent with the formula. It is shown that a key element for removing this discrepancy lies in a careful examination of the properties of the entrance loss coefficient. In particular, measurements of the entrance loss coefficient at small widths done with a physical model of the glottis (M5) show that this coefficient varies inversely with the glottal width. A numerical solution of the time-dependent equations of the surface wave model shows that adding a supraglottal vocal tract lowers the phonation threshold pressure by an amount approximately consistent with Chan and Titze's experiments.     

Sensitivity of vocal fold vibratory modes to their three-layer structure: implications for computational modeling of phonation

The sensitivity of the eigenmodes and eigenfrequencies of the human vocal fold to its three-layer structure is studied using finite-element modeling. The study covers a variety of three-dimensional vocal fold models ranging from an idealized, longitudinally uniform structure to a physiologically more realistic, longitudinally varying structure. Geometric parameters including the thickness of the ligament and cover layers as well as the ligament length are varied systematically. The results indicate that vocal fold vibratory modes are quite insensitive to the longitudinal variation in the thickness of the three layers as well as the variation in ligament length. However, significant overall changes in thickness of each layer can produce noticeable changes in these modes. The implications of these findings on computational modeling of phonation are discussed.     

Comments on single-mass models of vocal fold vibration

Proposed mechanisms for single-mass oscillation in the vocal tract are examined critically. There are two areas that distinguish single-mass models: in the sophistication of the air flow modeling near the oscillator and whether or not oscillation depends on acoustic feedback. Two recent models that do not depend on acoustic feedback are examined in detail. One model that depends on changing flow separation points is extended with approximate calculations.     

Infraglottic aspect of canine vocal fold vibration: Effect of increase of mean airflow rate and lengthening of vocal fold

The mucosal upheaval (MU), where the mucosal wave starts and propagates upward, appears only when the vocal fold vibrates. The location of the MU histologically and the effect of changes in mean air flow rate (MFR) and vocal fold length on occurrence of the MU were studied in twelve excised canine larynges. The lower surface of the vocal fold was marked to serve as a landmark for subsequent study. Cricothyroid approximation was performed to lengthen the vocal fold. After taking high-speed pictures or recording stroboscopic images from the tracheal side, a small cut wound was made at the mark. This wound served to compare the position of the MU with the histologically identified location of the mark. The larynx was then sectioned in the frontal plane. Before lengthening the vocal fold, the MU occurred on the area where the lamina propria became thinner and where the muscular layer neared the epithelial layer. After lengthening the vocal fold, the MU actually shifted medially compared with its original position. The subglottic area surrounded by the bilateral MUs became longer and thinner. Whether or not complete glottal closure during a vibratory cycle was achieved did not alter these findings. In contrast, with a fixed vocal fold length the MU appeared more laterally as MFR increased, but, based on the relation with the mark, its location on the vocal fold did not change from its original position before increase of MFR.     

Videokymography: High-speed line scanning of vocal fold vibration

A digital technique for high-speed visualization of vibration, called videokymography, was developed and applied to the vocal folds. The system uses a modified video camera able to work in two modes: high-speed (nearly 8,000 images/s) and standard (50 images/s in CCIR norm). In the high-speed mode, the camera selects one active horizontal line (transverse to the glottis) from the whole laryngeal image. The successive line images are presented in real time on a commercial TV monitor, filling each video frame from top to bottom. The system makes it possible to observe left-right asymmetries, open quotient, propagation of mucosal waves, movement of the upper and, in the closing phase, the lower margins of the vocal folds, etc. The technique is suitable for further processing and quantification of recorded vibration.     

Effect of artificially lengthened vocal tract on vocal fold oscillation's fundamental frequency.

The fundamental frequency of vocal fold oscillation (F(0)) is controlled by laryngeal mechanics and aerodynamic properties. F(0) change per unit change of transglottal pressure (dF/dP) using a shutter valve has been studied and found to have nonlinear, V-shaped relationship with F(0). On the other hand, the vocal tract is also known to affect vocal fold oscillation. This study examined the effect of artificially lengthened vocal tract length on dF/dP. dF/dP was measured in six men using two mouthpieces of different lengths. Results: The dF/dP graph for the longer vocal tract was shifted leftward relative to the shorter one. Conclusion: Using the one-mass model, the nadir of the "V" on the dF/dP graph was strongly influenced by the resonance around the first formant frequency. However, a more precise model is needed to account for the effects of viscosity and turbulence.     

The effect of topical anesthesia on vocal fold motion

The objective of this study was to determine if topical anesthesia to the larynx and pharynx affects vocal fold motion during dynamic voice evaluation with transnasal flexible endoscopy. Transnasal dynamic laryngeal examinations of 10 patients with no voice complaints were evaluated by five blinded fellowship-trained laryngologists. Each patient was examined before and after application of topical anesthetic. Reviewers rated briskness of right and left vocal fold movement and longitudinal tension on a visual analogue scale. Statistical comparisons were made between individual subject scores before and after anesthetic application. Inter-rater reliability was also assessed. No statistical difference was observed between subject scores before and after anesthetic application. Average intraclass correlation coefficients were 0.643 and 0.591 for pre- and postanesthesia scores, respectively. Application of topical anesthesia to the larynx and pharynx does not affect vocal fold motion.     

A simple-shear rheometer for linear viscoelastic characterization of vocal fold tissues at phonatory frequencies

Previous studies reporting the linear viscoelastic shear properties of the human vocal fold cover or mucosa have been based on torsional rheometry, with measurements limited to low audio frequencies, up to around 80 Hz. This paper describes the design and validation of a custom-built, controlled-strain, linear, simple-shear rheometer system capable of direct empirical measurements of viscoelastic shear properties at phonatory frequencies. A tissue specimen was subjected to simple shear between two parallel, rigid acrylic plates, with a linear motor creating a translational sinusoidal displacement of the specimen via the upper plate, and the lower plate transmitting the harmonic shear force resulting from the viscoelastic response of the specimen. The displacement of the specimen was measured by a linear variable differential transformer whereas the shear force was detected by a piezoelectric transducer. The frequency response characteristics of these system components were assessed by vibration experiments with accelerometers. Measurements of the viscoelastic shear moduli (G' and G") of a standard ANSI S2.21 polyurethane material and those of human vocal fold cover specimens were made, along with estimation of the system signal and noise levels. Preliminary results showed that the rheometer can provide valid and reliable rheometric data of vocal fold lamina propria specimens at frequencies of up to around 250 Hz, well into the phonatory range.     

Nonlinear behavior of vocal fold vibration: The role of coupling between the vocal folds

Coupling between the vocal folds is one of the nonlinear mechanisms allowing regulation and synchronization of mucosal vibration. The purpose of this study was to establish that modulations such as diplophonia and abnormalities observed in vocal signals that may be observed in some cases of laryngeal pathology can be considered as nonlinear behavior due to the persistence of some physical interaction (coupling). An experimental model using excised porcine larynx was designed to create tension asymmetry between the vocal folds and to obtain vocal signals with modulations. Signals were analyzed by spectral analysis and the phase portrait method. Results were compared with computer-generated synthetic signals corresponding to nonlinear combinations of sinusoid signals. Under these conditions, evidence of nonlinear behavior was detected in 85% of experimental signals. These findings were interpreted as a demonstration of vocal fold interaction. Based on these findings, the authors conclude that (1) coupling must be taken into account in physical models of laryngeal physiology, and that (2) methods of nonlinear dynamics may be used for objective voice analysis.     

Dependence of phonation threshold pressure on vocal tract acoustics and vocal fold tissue mechanics

Analytical and computer simulation studies have shown that the acoustic impedance of the vocal tract as well as the viscoelastic properties of vocal fold tissues are critical for determining the dynamics and the energy transfer mechanism of vocal fold oscillation. In the present study, a linear, small-amplitude oscillation theory was revised by taking into account the propagation of a mucosal wave and the inertive reactance (inertance) of the supraglottal vocal tract as the major energy transfer mechanisms for flow-induced self-oscillation of the vocal fold. Specifically, analytical results predicted that phonation threshold pressure (Pth) increases with the viscous shear properties of the vocal fold, but decreases with vocal tract inertance. This theory was empirically tested using a physical model of the larynx, where biological materials (fat, hyaluronic acid, and fibronectin) were implanted into the vocal fold cover to investigate the effect of vocal fold tissue viscoelasticity on Pth. A uniform-tube supraglottal vocal tract was also introduced to examine the effect of vocal tract inertance on Pth. Results showed that Pth decreased with the inertive impedance of the vocal tract and increased with the viscous shear modulus (G") or dynamic viscosity (eta') of the vocal fold cover, consistent with theoretical predictions. These findings supported the potential biomechanical benefits of hyaluronic acid as a surgical bioimplant for repairing voice disorders involving the superficial layer of the lamina propria, such as scarring, sulcus vocalis, atrophy, and Reinke's edema.