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.

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 &#176;C minimizes the potential of tissue degradation and alteration of biomechanical properties, and allows performing the experiments at any convenient time.
<ol>
	<li>Defrosting the larynx
	<ol>
		<li>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 &#176;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.</li>
	</ol>
	</li>
	<li>Cleaning the larynx
	<ol>
		<li>After the larynx is defrosted, remove it from the bag and clean it thoroughly with saline solution (0.9% NaCl).</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Exposure of the thyroid and cricoid cartilages
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Mid-sagittal cut through the thyroid cartilage
	<ol>
		<li>Make an initial vertical cut through the anterior part of the thyroid cartilage.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Cutting the cricoid cartilage
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Removal of one vocal fold, creating an L-shaped incision in the larynx
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Refinement of the cut through the thyroid cartilage
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg" /> 
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. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

2. Hemi-larynx Experiment
<ol>
	<li>Hemi-laryngeal setup
	<ol>
		<li>Use an air-supply tube which delivers warmed and humidified air into the larynx.</li>
		<li>Construct two perpendicularly arranged transparent plates as a substitution for removed laryngeal parts.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg" />
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. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Mounting the hemi-larynx
	<ol>
		<li>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.</li>
		<li>Fasten the trachea with a plastic tightening strap or a hose clamp.&#160;</li>
		<li>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.</li>
		<li>Attach the transparent plates.</li>
	</ol>
	</li>
	<li>Stabilization of the thyroid cartilage, adduction of the vocal fold using prongs
	<ol>
		<li>Use the prongs to adduct the vocal fold to the plate and stabilize the thyroid cartilage.</li>
		<li>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.</li>
		<li>Seal eventually occurring gaps by adding more fixative cream.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The hemi-larynx preparation shares the advantages of the &#34;conventional&#34; (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 %, yet still being within realistic ranges16. The major disadvantage of the full excised larynx approach, i.e., the lack of visibility of the vocal fold surface along the superior-inferior (caudo-cranial) dimension, is overcome in the hemi-larynx setup by providing a side view of the vibrating vocal fold. The hemi-larynx setup thus allows assessment of vocal fold motion in multiple dimensions, which is crucial when trying to understand the finer details of the biophysical sound generation mechanism in humans and nonhuman mammals.
Here, several exemplary applications of the hemi-larynx setup have been demonstrated. The documentation of vocal fold vibration from two camera angles allows further qualitative and quantitative data analysis. The kymographic glottal motion analysis in the vertical direction, newly introduced in this paper, allows reconstruction of the temporal geometric variations of the glottis along a selected position along the antero-posterior (dorso-ventral) glottal axis. When repeating this analysis for several points equidistantly spaced along the glottal axis, the entire glottal motion could be reconstructed. Note that this approach provides comparable but not identical results as compared to assessment of vocal fold motion by marking and tracking individual &#34;fleshpoints&#34; in the vocal fold tissue (also on points not forming the glottis), e.g., with micro-sutures20 or silicon carbide particles5,40. Precise knowledge about the time-varying glottal geometry in three dimensions is crucial to further investigate details of the glottal airflow and its interaction with the vibrating laryngeal tissue. For example, computational models of self-sustaining vocal fold vibration could be improved as more empirical data concerning the point of airflow jet separation 41,42,43,44,45,46,47,48 become available.
As illustrated in Figure 5, the hemi-larynx preparation enables assessment of the vocal fold contact area (VFCA) during self-sustained vocal fold vibration. For one, knowledge of the time-varying relative magnitude of VFCA is useful to validate the results from electroglottographic measurements27, as EGG is a widely-used method for non-invasive assessment of vocal fold vibration in vivo. Furthermore, measurement of the exact VFCA geometry and its variation over time might prove to be crucial for better understanding the notion of vocal fold contact depth 49 and its potential relation to the speed of the so-called mucosal wave50,51,52,53. There, an airflow-driven travelling wave occurs within the surface cover layer of the vocal fold tissue. This wave moves initially along with the trans-glottal airflow from the inferior to the superior vocal fold edge, and then it propagates laterally across the upper vocal fold surface once every oscillatory cycle54.
All things considered, the hemi-larynx approach is a powerful, yet not widely used constituent of the currently available arsenal of empirical methods for basic voice science. Here, a tutorial for creating a hemi-larynx preparation is presented, and some potential future applications are discussed. The given instructions may help improving the repeatability of experiments across different labs, thus providing voice scientists with the potential to better understand the biomechanics of voice production.

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 (&#34;chest&#34; vs. &#34;falsetto&#34; 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 &#34;conventional&#34; 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.

<table><tbody><tr><td>Surgical blades</td><td>Surgeon</td><td>Jai Surgical Ltd., New Delhi, India</td><td></td></tr><tr><td>Saw</td><td>Hand saw (Lux, 150 mm length)</td><td>Lux, Wermelskirchen, Germany</td><td></td></tr><tr><td>Thermometer</td><td>Testo 922</td><td>Testo Ltd., Hampshire, UK</td><td>K-type Probe, Operating temperature -20 to +50 &amp;deg;C</td></tr><tr><td>Autoclave bags</td><td>Autoclave bags</td><td>vwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic</td><td></td></tr><tr><td>Conductive glass plates</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>Fixative cream</td><td>Denture fixative cream</td><td>Blend-a-dent Natural</td><td></td></tr><tr><td>Prongs and fastening system</td><td>Customized Kanya Al eloxed profiles</td><td>Distributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.;</td><td>&amp;nbsp;Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs</td></tr><tr><td>Mounting tube</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics,&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>LED Light</td><td>Verbatim 52204 LED Lamp</td><td>Mitsubishi Chemical Holdings Corporation, Tokyo, Japan</td><td></td></tr><tr><td>Camera</td><td>Canon EOS1100D</td><td>Canon Inc.</td><td>18-55 mm lens</td></tr><tr><td>Airpump</td><td>Resun LP100</td><td>Resun</td><td></td></tr><tr><td>Strobe light</td><td>ELMED Helio-Strob micro2</td><td>ELMED Dr. Ing. Mense GmbH, Heiligenhaus, Germany</td><td></td></tr><tr><td>Humidifier</td><td>Custom made</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td></td></tr><tr><td>Subglottic tract</td><td>Custom made adjustable subglottic tract</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td>Hampala, 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</td></tr></tbody></table>

hemi-laryngeal setup for studying vocal fold vibration in three dimensions

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.

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-larynx vocal fold was documented from the top and from the side with two synchronized high-speed video (HSV) cameras operated at 6,000 frame/s, complemented by time-synchronous recordings of acoustic and electroglottographic (see below) data sampled at 44.1 kHz. More information on the data acquisition setup including a list of equipment used can be found in previous publications by this group of authors 27,28. Footage from these HSV recordings is shown in the accompanying video. Still images, extracted at representative moments within the vibratory cycle, are shown in Figure 3. The top view (upper half of each panel) shows medio-lateral vocal fold movement, indicating an open glottis in Figure 3A, allowing glottal air flow, whilst in Figure 3B-D the glottis is closed (the vocal fold is in complete contact with the vertical glass plate), thus arresting the glottal air flow. The side view (lower half of each panel) in Figures 3B-D suggests a varying degree of vocal fold contact against the glass plate, as well as a varying geometry and vertical location of that contact.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55303/55303fig3.jpg" /> 
Figure 3: Hemi-larynx vocal fold vibration. (A-D) Still images from high-speed video footage from the top (upper half of each panel) and the side view cameras (lower half of each panel), extracted at representative points within the vibratory cycle. Note the absence of vocal fold contact in (A), and the varying (both in area, shape, and position) vocal fold contact in (B-D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Kymographic glottal motion analysis
Quantitative glottal motion analysis is illustrated in Figure 4. The glottis is the variable opening between the (vibrating) vocal folds29, created by their deflections during self-sustained oscillation. State of the art analysis of top view HSV footage allows tracing the lateral deflections of the vocal folds30,31. The hemi-larynx preparation described here adds the facility to assess also the vertical (caudo-cranial) aspects of vocal fold vibration.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55303/55303fig4.jpg" /> 
Figure 4: Kymographic glottal motion analysis. 
(A) and (B) Video stills showing top and side views of the hemi-larynx, taken from high-speed video (HSV) recordings at 6,000 frames/s. The yellow vertical lines indicate the kymographic scan line position for the kymograms shown in panels C and E for the top view, and panels D and F for the side view. (C) and (D) Digital kymograms extracted from the HSV footage of the top and the side view, respectively. (E) The time-varying lateral displacement of the vocal fold extracted from the kymogram and traced with a line (short dashes). (F) The time-varying deflections of the inferior and superior edges of the vocal fold, assessed from the kymogram and traced with a dashed and a dotted line, respectively. (G) Synoptic depiction of the time-varying glottal structures: Lateral vocal fold deflection (&#34;top&#34;, pale violet), and vertical deflection of the superior (&#34;side sup.&#34;, dark red) and inferior (&#34;side inf.&#34;, dark green) vocal fold edges extracted from the kymograms shown in panels E and F. (H) Motion speeds derived from the glottal structure displacement data shown in panel G. (I) and (J) Glottal motion reconstruction derived from the displacement data of the superior and inferior vocal fold margins shown in panel G. The arrows indicate the direction of the rotational movement. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Two digital kymograms were generated from top and side view HSV data (Figures 4C and 4D). In a digital kymogram (DKG)32,33,34,35, the pixel data from a single line (typically at the point of maximum vocal fold vibratory amplitude), taken from a number of consecutive high-speed video frames, are concatenated to form a temporal axis on the abscissa. The time-varying displacement of the structures covered by the DKG scan line is visible on the ordinate. In the example shown in Figure 4C-F, the DKG scan line positions of the top and side view were selected halfway along the antero-posterior (ventro-dorsal) dimension of the vocal fold, utilizing the approach described by Hampala et al., Eq. 127.
The lateral and caudo-cranial deflections of the glottis, delineated by the inferior and superior vocal fold edges, were traced within the DKG data (Figures 4E and 4F) and expressed in metric units based on the video frame rate and calibration information embedded in the videos (Figure 4G and H). A reconstruction of the two-dimensional (lateral and vertical) glottal motion at the middle of the vocal fold (i.e., the place of maximum vibratory amplitude) over three complete glottal cycles is shown in Figure 4E and F. During the majority of the glottal cycle, the vocal fold was in contact with the glass plate (representing glottal closure), but with varying contact depth. During the open phase (i.e., when the vocal fold is not in contact with the glass plate), the traces of the inferior and the superior vocal fold edge fuse, and they exhibit a complex cyclic motion pattern, in partial agreement with results from other studies 5,20,36,37 (the motion pattern found in humans tends to be more elliptical than that of the red deer specimen investigated here). Interestingly, the vertical displacement reached a vibratory amplitude of about 10 mm, i.e., almost an order of magnitude larger than what was found in humans.
Assessment of vocal fold contact area
Electroglottography (EGG)38 is a widely used non-invasive method for measuring changes in relative vocal fold contact area (VFCA) during phonation. A low intensity, high-frequency current is passed between two electrodes placed at vocal fold level on each side of the larynx. The admittance variations resulting from vocal fold (de)contacting during laryngeal sound production are largely proportional to the time-varying relative vocal fold contact area39. The EGG signal is assumed to be a reliable physiological correlate of vocal fold vibration, reflecting the fundamental frequency and the oscillatory regime (irregular or periodic, including bifurcations). Despite its broad application, the possible direct relation between the VFCA and the EGG waveform has, until recently, only been tested in a single study17, suggesting an approximately linear relationship between VFCA and the EGG signal magnitude. However, flow-induced vocal fold vibration was not investigated in that study. Therefore, a rigorous empirical evaluation of EGG as a measure of relative VFCA under proper physiological conditions was therefore still needed.
In addressing this issue, this group of authors has recently investigated three red deer larynges in an excised hemi-larynx preparation utilizing a conducting glass plate27. The time varying contact between the vocal fold and the glass plate was monitored by high-speed video recordings made in the sagittal plane at 6000 fps, synchronized with the EGG signal with an accuracy of &#177; 0.167 ms. Representative results from that study are shown in Figure 5, indicating an average to good agreement between the EGG signal and VFCA &#8211; see reference27 for details).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55303/55303fig5.jpg" /> 
Figure 5: Comparison of vocal fold contact area (VFCA) and electroglottographic (EGG) waveform. (A-D) Video stills from high-speed video data showing the side view of a red deer hemi-larynx at four instants within a glottal cycle. The manually assessed vocal fold contact area (i.e., the area where the vocal fold was in contact with the vertical glass plate in the hemi-larynx setup) is superimposed in cyan. (E) Comparison of normalized EGG and VFCA data for the vocal fold contact phase of one glottal cycle. The VFCA data stemmed from assessment of vocal fold contact area (counted in pixels) over the glottal cycle. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55303/55303fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions at JoVE.com

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

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.

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

hemi-laryngeal-setup-for-studying-vocal-fold-vibration-three

Nanyang Technological University

Research

JoVE Journal

Biology

10.9K Views.  Palacky University Olomouc. Deze paper introduceert een protocol voor de bereiding van hemi-strottenhoofd specimens te vergemakkelijken van een multi-dimensionale weergave van vocale vouw trillingen, om te onderzoeken van verschillende biofysische aspecten van de productie van de stem in de mensen en niet-menselijke zoogdieren.

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

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Bioengineering

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

3D Printing of Multilayered Silicone Vocal Fold Models

Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal fold models results in models with undesirable properties, such as stickiness and reproducibility issues. Those vocal fold models are prone to rapid aging, leading to poor comparability across different measurements. In this study, we propose a modification to the manufacturing process by changing the order of layering the silicone material, which leads to the production of non-sticky and highly consistent vocal fold models. We also compare a model produced using this method with a conventionally manufactured vocal fold model that is adversely affected by its sticky surface. We detail the manufacturing process and characterize the properties of the models for potential applications. The outcomes of the study demonstrate the efficacy of the modified fabrication method, highlighting the superior qualities of our non-sticky vocal fold models.&#160;The findings contribute to the development of realistic and reliable vocal fold models for research and clinical applications.

Author Spotlight: Advancements in the Fabrication of Synthetic Vocal Fold Models for Phonetic and Robotic Applications

This study demonstrates the manufacturing of non-sticky and super-soft vocal fold models by introducing a specific way to create the vocal fold layers, providing a detailed description of the manufacturing procedure, and characterizing the properties of the models.

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal ...

Engineering

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Neuroscience

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

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

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