## 3 Proof-of-Concept Using an Excised Whole-Mount Human Larynx Model  
### Materials and Methods  
A proof-of-concept experiment was conducted with an excised human larynx to simulate unilateral vocal fold phonation using airflow-driven oscillation at varying levels of subglottal pressure. After obtaining the tissue, the larynx was stored in a freezer at -80 \({}^{\circ}\)C and subsequently thawed for the current experiment. Freezing and thawing has been shown to preserve the visco-elastic properties of vocal fold tissue and allow for the assessment of biomechanics of phonation [20].  
Figure 2 displays the whole-mount setup of the excised cadaver larynx that was prepared by dissecting away supraglottal structures (hyoid, etc., such that there was no supraglottal tract), suturing the ventricular folds laterally, and removing some of the superior aspect of the thyroid laminae to provide exposure of the true vocal folds. Inferiorly, the trachea was cut to a length of approximately 5 cm, and the specimen was connected to an air supply via a cylindrical tube 1.5 m in length and inner diameter of 20 mm. The larynx was mounted in a holding device that used 2 pairs of corkscrew-tipped anchoring arms to rigidly fixate the thyroid and cricoid cartilages. The arytenoid cartilages were sutured together in adduction using a single stitch passed through the bodies of the cartilages. Airflow from a medical air supply was sent through a Hudson RCI ConchaTherm III device (Teleflex, Morrisville, NC, USA) that warmed and humidified the air to 37 \({}^{\circ}\)C before directing the air stream through the trachea to produce self-sustained vocal fold oscillations. A pneumatic pressure gauge regulated the driving pressure of the air stream, and a secondary pressure transducer acted as a reference subglottal pressure recording from approximately 6 cm below the vocal folds.  
Figure 2: Whole-mount cadaver excised larynx preparation with ISP probe sensors measuring intraglottal pressure (IGP) and subglottal pressure (SGP) signals. An acoustic microphone was placed 15 cm from the glottis, and an accelerometer recorded the vibrations of the anterior, external tracheal wall of the specimen.  
A right-handed ISP probe was positioned such that the intraglottal pressure signal measured the vocal fold collision pressure from the right vocal fold (see Figure 2). The ISP probe was placed intraglottally and pressed against the left vocal fold, effectively creating a scenario in which the left vocal fold did not vibrate when the airflow was turned on. Signals were recorded simultaneously to a data acquisition system (Digidata 1440, Molecular Devices, Sunnyvale, CA, USA) from five sensors: (1) an acoustic microphone, (2) the ISP probe's intraglottal pressure sensor, (3) the ISP probe's subglottal pressure sensor, (4) the secondary subglottal pressure sensor placed within the trachea in the path of the airflow (Model MPX2010 GP; Motorola, Schaumburg, IL, USA), and (5) a high-bandwidth accelerometer (model BU-27135; Knowles Corp., Itasca, IL, USA). Lowpass anti-aliasing filtering was applied on all channels with a 3-dB cutoff frequency of 8 kHz (CyberAmp 380, Molecular Devices) prior to digitization at a 20 kHz sampling rate per channel. This setup enabled the measurement of vocal fold collision pressures at varying levels of subglottal pressure. Future experiments may continue to investigate effects of fundamental frequency, and vocal fold adduction/abduction forces.  
The use of human tissue for experimental study was approved by the Partners HealthCare System Institutional Review Board at Massachusetts General Hospital.