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Method Article

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

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DOI:

10.3791/55303

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November 25th, 2017

 ,  ,  ,  , 
1Voice Research Lab, Department of Biophysics, Faculty of Science, Palacky University Olomouc, 2Laboratory of Bio-Acoustics, Dept. of Cognitive Biology, University of Vienna, 3ENES Lab, NEURO-PSI,CNRS UMR 9197, Université Lyon/Saint-Etienne, France

In This Article

Summary

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

Abstract

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.

Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

Introduction

Voice is typically created by vibrating laryngeal tissue (mainly the vocal folds), which converts a steady airflow, supplied by the lungs, into a sequence of airflow pulses. The acoustic pressure waveform (i.e., the primary sound) emerging from this sequence of flow pulses acoustically excites the vocal tract which filters them, and the resulting sound is radiated from the mouth and (to a certain degree) from the nose1. The spectral composition of the generated sound is largely influenced by the quality of vocal fold vibration, governed by laryngeal biomechanics and interactions with the tracheal airflow2. Both in a clinical and a research context, documentation and assessment of vocal fold vibration is thus of foremost interest when studying voice production.

In humans, direct endoscopic investigation of the larynx during sound production in vivo is challenging, and it is virtually impossible in nonhuman mammals, given current technological means. Therefore, and in order to guarantee carefully controlled physical and/or physiological experimental boundary conditions, the use of excised larynges3,4 is in many cases an adequate substitution for investigation of in vivo voice production mechanisms.

Vocal fold vibration is a complex three-dimensional phenomenon5. While conventional investigation methods like laryngeal endoscopy (in vivo) or excised larynx preparations typically provide only a superior view of the vibrating vocal folds6, they do not allow for complete three-dimensional analysis of vocal fold motion. In particular, in the superior view the lower (caudal) margins of the vocal folds are invisible during a major portion of the vibratory cycle. This is due to the phase delay between the inferior (caudal) and the superior (cranial) edge of the vocal folds, a phenomenon which is typically seen during vocal fold oscillation5. As direct empirical evidence for backing up findings from mathematical and physical models is scarce, knowledge of the geometry and motion of the lower vocal fold edge7, and thus the geometry of the subglottal channel8,9,10 is crucial for better understanding the interaction between laryngeal airflow, vocal fold tissue, and the resulting forces and pressures11,12. Another aspect of vocal fold vibration that is hidden from the customary superior view is the vertical (caudo-cranial) depth of the contact between the two vocal folds. The vertical contact depth is related to the vertical thickness of the vocal folds, which is a potential indicator of the vocal register used in singing ("chest" vs. "falsetto" register)13,14.

In order to overcome the shortcomings of conventional (full) excised larynx preparations, a so-called hemi-larynx setup can be utilized, where one half of the larynx is removed, thus facilitating the assessment of the vibratory characteristics of the remaining vocal fold in three dimensions. Surprisingly, since the introduction of this setup in the 1960s15 and an initial validation of the concept in 199316, not many laboratories have performed experiments with this promising experimental approach17,18,19,20,21,22,23. An explanation for this might be found in the difficulties of creating a viable hemi-larynx preparation. While the conventional excised (full) larynx preparation is well documented4, no such in-depth instructions are as yet available for creating a hemi-larynx setup. It is therefore the purpose of this paper to provide a tutorial for establishing a reliably reproducible hemi-larynx setup, supplemented by experimental results from red deer specimens.

A hemi-larynx setup shares many features with a "conventional" excised larynx setup, such as measurement equipment, high-speed or other imaging technology to adequately document the vibrations of the laryngeal structures during sound generation, or proper supply of heated, humidified air. These general setup considerations are described in detail in both a book chapter4 and a technical report from the National Center of Voice and Speech24. Reiteration of these instructions would be beyond the scope of this manuscript. Here, only the specialized directives for generating a hemi-larynx setup are presented.

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Protocol

The animal specimens analyzed in this paper were treated in accordance with the standard ethical requirements of the Palacky University in Olomouc, Czech Republic. They stem from red deer living wildly in forests, which were hunted by the Czech Army Forest Service during a regular hunting season.

1. Preparation of the Hemi-larynx Specimen

Note: Only properly prepared specimens should be used, as indicated in4 . Quick freezing of the larynx25 immediately after excision and storage at -80 °C minimizes the potential of tissue degradation and alteration of biomechanical properties, and allows performing the experiments at any convenient time.

  1. Defrosting the larynx
    1. Insert the frozen larynx into two autoclave bags or any other plastic bags with waterproof sealing. Seal the bags and put them into a water bath heated to 30 °C until the larynx is completely defrosted. The duration required ranges from a few hours to more than a day, depending on larynx size and freezing temperature.
  2. Cleaning the larynx
    1. After the larynx is defrosted, remove it from the bag and clean it thoroughly with saline solution (0.9% NaCl).
    2. Carefully remove superfluous tissue as applicable (i.e. external neck muscles, hyoid bone etc.) without damaging the main laryngeal structures, and shorten the trachea to a length adequate for mounting the larynx onto an air supply tube (usually ca. 4-5 cm).
    3. Check the laryngeal tissue for potential tissue anomalies, such as wounds, organic deformations, or cracks potentially occurring from the freezing process, which could make the larynx unsuitable for the experiment.
  3. Exposure of the thyroid and cricoid cartilages
    1. Remove parts of the external laryngeal muscle tissue around the thyroid and cricoid cartilage using a scalpel, thus exposing the cartilages in preparation for the mid-sagittal cut creating the hemi-larynx. This preparation stage is depicted in Figure 1A and 1B.
  4. Mid-sagittal cut through the thyroid cartilage
    1. Make an initial vertical cut through the anterior part of the thyroid cartilage.
    2. Carefully place the cut slightly more onto the side that is about to be removed, in order not to damage the vocal fold that needs to remain preserved. If possible, use a scalpel for cutting. If the cartilage is ossified, use a small saw.
  5. Cutting the cricoid cartilage
    1. Lead the cut vertically (inferiorly) from in between the arytenoid cartilages and then through the cricoid cartilage to an approximately horizontal level of the inferior thyroid notch.
  6. Removal of one vocal fold, creating an L-shaped incision in the larynx
    1. Make a horizontal cut starting from the inferior end of the previously made vertical cut in the cricoid cartilage, and lead the new cut towards the inferior thyroid notch. Anteriorly fold the side of the larynx that is going to be removed.
    2. Make a vertical cut through the soft tissue on the inner side of the thyroid cartilage - be careful while leading the cut in between the anterior attachment of the vocal folds to the thyroid cartilage, thus avoiding damage to the vocal fold.
  7. Refinement of the cut through the thyroid cartilage
    1. Use a scalpel, a saw, or a file, in order to apply a precisely straight cut in the thyroid cartilage, and get as close as possible to the anterior part of the previously inspected vocal fold.
    2. Remove also a small part of the posterior thyroid cartilage, in order to create space for inserting the prong for adducting the arytenoid cartilage and thus the vocal fold (see below). This preparation stage is depicted in Figure 1C and 1D.
      Note: Depending on the research question, full exposition of the whole vocal fold may be needed to enable its visibility from above. In such a case, the structures above the (true) vocal fold (i.e., the ventricular or vestibular fold, as applicable given the anatomy of the specimen) should be removed. In some specimens the inner soft laryngeal tissue above the vocal folds might lose its connection with the thyroid cartilage and interferes with the vocal fold during vibration, potentially causing spurious (mostly irregular) oscillatory patterns. In such a case careful removal of that tissue is inevitable.

Laryngeal anatomy diagram showing epiglottis, thyroid, cricoid cartilages, vocal folds, arytenoids.
Figure 1: Hemi-larynx preparation and mounting. (A) and (B) Cleaned larynx specimen, medial and posterior view, before removal of left vocal fold; (C) and (D) Prepared hemi-larynx with L-shaped incision (left vocal fold removed), medial and posterior view. Please click here to view a larger version of this figure.

2. Hemi-larynx Experiment

  1. Hemi-laryngeal setup
    1. Use an air-supply tube which delivers warmed and humidified air into the larynx.
    2. Construct two perpendicularly arranged transparent plates as a substitution for removed laryngeal parts.
    3. Use prongs 4 for increasing the stability of the larynx and creating a proper pre-phonatory larynx configuration by adducting the remaining vocal fold to the vertical glass plate (see Figure 2A).
      Note: Theoretically, the vocal folds might also be adducted by sutures and weights on a pulley-lever system 26 . However, such an approach has, to the best knowledge of these authors, not yet been attempted for a hemilarynx preparation.

Static equilibrium experiment setup with glass plates and adduction prongs, anterior view.

Figure 2: Hemi-larynx setup. (A) Supporting structures: air supply tube, L-shaped glass plate arrangement, adduction prongs. (B) Mounted hemi-larynx preparation with adduction prongs. (C) and (D) Close-ups of hemi-larynx-preparation, viewed from the side and from the top, respectively. Please click here to view a larger version of this figure.

  1. Mounting the hemi-larynx
    1. Cover the air supply tube with denture fixative cream and mount the larynx using the remaining part of its trachea. The fixative cream works as an adhesive and closes potential gaps, thus creating an air-tight seal.
    2. Fasten the trachea with a plastic tightening strap or a hose clamp. 
    3. Also cover the edges of the cut through the thyroid cartilage with the fixative cream, while avoiding spreading fixative cream on the vocal fold or the inner soft laryngeal tissues.
    4. Attach the transparent plates.
  2. Stabilization of the thyroid cartilage, adduction of the vocal fold using prongs
    1. Use the prongs to adduct the vocal fold to the plate and stabilize the thyroid cartilage.
    2. After the fixative cream has set, apply the airflow in order to establish vocal fold oscillation and check for possible leaks between the hemi-larynx and the glass plates.
    3. Seal eventually occurring gaps by adding more fixative cream.

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Results

Illustrations of the hemi-larynx preparation and its mounting on the air supply tube, as referenced in the previous section, are provided in Figure 1 and Figure 2, respectively.

Documentation of vocal fold vibration from two camera angles

Airflow-induced self-sustaining oscillation of the hemi...

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Discussion

The hemi-larynx preparation shares the advantages of the "conventional" (full) excised larynx setup: In such an experimental approach, physical and physiological boundary conditions and parameters (such as subglottal pressure or vocal fold elongation) can be controlled fairly well. The behavior of the hemilarynx is homologous to that of a full larynx with a perfect lateral symmetry, with the exception that magnitudes of some parameters (e.g., air flow rate, sound pressure) are reduced by approximately 50...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by an APART grant of the Austrian Academy of Sciences (CTH), the Technology Agency of the Czech Republic project no. TA04010877 (CTH, VH and JGS), and the Czech Science Foundation (GACR) project no GA16-01246S (to JGS). We thank W. Tecumseh Fitch for his suggestion to use denture fixative cream, and Ing. P. Liska from the Czech Army Forest Service for his help in acquiring the excised deer larynges.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Surgical bladesSurgeonJai Surgical Ltd., New Delhi, India
SawHand saw (Lux, 150 mm length)Lux, Wermelskirchen, Germany
ThermometerTesto 922Testo Ltd., Hampshire, UKK-type Probe, Operating temperature -20 to +50 °C
Autoclave bagsAutoclave bagsvwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic
Conductive glass platesCustom madeUPOL - Joint laboratory of Optics
Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.
Fixative creamDenture fixative creamBlend-a-dent Natural
Prongs and fastening systemCustomized Kanya Al eloxed profilesDistributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.; Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs
Mounting tubeCustom madeUPOL - Joint laboratory of Optics,
Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.
LED LightVerbatim 52204 LED LampMitsubishi Chemical Holdings Corporation, Tokyo, Japan
CameraCanon EOS1100DCanon Inc.18-55 mm lens
AirpumpResun LP100Resun
Strobe lightELMED Helio-Strob micro2ELMED Dr. Ing. Mense GmbH, Heiligenhaus, Germany
HumidifierCustom madeVoice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic
Subglottic tractCustom made adjustable subglottic tractVoice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republicHampala, V., Svec, Jan, Schovanek, P., and Mandat, D. Uzitny vzor c. 25585: Model subglotickeho traktu. [Utility model no. 25585: Model of subglottal tract] (In Czech) Soukup, P. 2013-27834(CZ 25505 U1), 1-7. 24-6-2013. Praha, Urad prumysloveho vlastnictvi

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Hemi larynx SetupHigh speed VideoGlottal Area MeasurementElectroglottography ValidationChemographic Glottal AnalysisLaryngeal Tissue PreparationAir Supply MountingSynchronized Camera ImagingThree dimensional Motion Analysis
Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions
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