The authors acknowledge Kendrea Focht, CScD, CCC-SLP, and the Evelyn Trammell Institute for Voice and Swallowing at the Medical University of South Carolina, for sharing MBS imaging files used to demonstrate this methodology. These MBS data were collected through extramural support funded by Grant Number TL1TR000061 (PI: Focht) from the National Center for Advancing Translational Sciences and by Grant Number 1K24DC12801 (PI: Martin-Harris) from the National Institute On Deafness And Other Communication Disorders, and intramural support from Mark and Evelyn Trammell Trust. These methods were originally developed by the principal investigator while supported by Grant Number F31DC011705 from the National Institute On Deafness And Other Communication Disorders. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Deafness And Other Communication Disorders or the National Institutes of Health.&#160;

1. Configuring a computer
<ol>
	<li>For Macintosh, download the following open source or freeware software: ImageJ, MacX Video Converter Free Edition (Mac), and QuickTime (see table of material/equipment).</li>
	<li>For a PC, download the following open source or freeware software: ImageJ, MPEG Streamclip (PC), and QuickTime (see table of material/equipment).</li>
</ol>
2. Preparing video clips
<ol>
	<li>File conversion. Convert raw video files into .mov for data collection in ImageJ. Note: usable videos should include the mandible, hard palate, C1-C4 vertebrae, larynx and pharynx in the field of view. ImageJ processing tools can improve image quality issues.
	<ol>
		<li>With a PC, use MPEG Streamclip, load the raw .avi video file via File &#62;&#62; Open Files and selecting the desired clip. Select File &#62;&#62; Export to QuickTime.</li>
		<li>With a Mac, use MacX Video Converter, click &#34;Add File&#34; and choose video(s) for conversion. Multiple videos can be converted at once using this application.</li>
		<li>In the Output Settings section, click the browse button and choose the desired location to save the converted file.</li>
		<li>Click on the &#34;to MOV&#34; tab. Make sure the Output Format under the &#34;Video Settings&#34; section is MOV. Click Start.</li>
	</ol>
	</li>
	<li>Editing clip length. Memory is limited with ImageJ. It is recommended that a file of each swallow be made.
	<ol>
		<li>Open MOV. Video file created in step 2.1 using QuickTime (PC or Mac).</li>
		<li>Identify 5 ml&#160;thin-liquid and pudding swallows by audio queues or by swallow sequence outlined by the MBS protocol used during data collection.</li>
		<li>Select Edit &#62;&#62; Trim and adjust the trimming bar so that the entire 5 ml&#160;Thin Fluid swallow is visualized. Click trim.</li>
		<li>Select File &#62;&#62; Export and create a file name that will be used to link subject data (gender, age, etiology, bolus type) to coordinate mapping results.</li>
		<li>Repeat for the pudding swallow (or for any other bolus of interest).</li>
	</ol>
	</li>
</ol>
3. De-identifying images
<ol>
	<li>If a file contains any personal health information (PHI) and needs to be de-identified, upload the file using Image J (See 4.1 - 4.2).</li>
	<li>Use the rectangle tool to frame the swallowing study to exclude PHI. Select Image &#62;&#62; Crop. Then Select File &#62;&#62; Save As &#62;&#62; QuickTime Movie.</li>
	<li>Configure the dialogue box as follows: Compression &#62;&#62; Sorenson 3; Quality &#62;&#62; Maximum, enter the appropriate frame rate (usually 30 fps).&#160;</li>
</ol>
4. Preparing to measure
<ol>
	<li>Open ImageJ. Click on the &#34;&#62;&#62;&#34; icon on the toolbar. Select &#62;&#62; Arrow Labeling Tools.</li>
	<li>Uploading images. Click on QuickTime icon. Select &#34;Open movie as a stack&#34; from the drop down menu and locate edited QuickTime clip. Image J will not open the entire swallow study due to memory limits (see step 2.2). Memory can be minimally expanded by selecting Edit &#62;&#62; Options &#62;&#62; Memory &#38; Threads.</li>
	<li>Processing images to improve image quality. Select Process &#62;&#62; Math &#62;&#62; Add. Check the preview box and adjust numbers to desired image quality. Reply yes to process the entire stack of images.</li>
	<li>Set measurements. Select Analyze &#62;&#62; Set Measurements from the ImageJ menu. At the dialogue box mark &#34;Stack Position&#34; and &#34;Invert Y coordinates&#34;. Unmark everything else.</li>
	<li>Select the multipoint tool from the toolbar. Click on anatomical landmarks in sequence (see 5.0)</li>
	<li>Using the multipoint tool.
	<ol>
		<li>Take a measurement of all points by selecting Analyze &#62;&#62; Measure from the menu or keyboard command+M (control+M for PCs).</li>
		<li>Remove all points with command+A (control+A for PCs).</li>
		<li>Remove single points by hovering over a point, then hold the command-option keys and click on the point to be removed.</li>
		<li>Move singular points by hovering over a point, clicking, dragging and dropping a point to a new location.</li>
		<li>Move all points together with arrow keys.</li>
	</ol>
	</li>
</ol>
5. Mapping Landmarks
<ol>
	<li>Begin at first frame and advance to a clear frame in the pre-oral phase. Observe position of the bolus on the anterior, superior margin of the tongue prior to initiation of oral transport of the swallow. Use this frame to set the first nine coordinates.</li>
	<li>Use the ImageJ multi-point tool to map the first nine coordinates at the following anatomical landmarks (see Figures&#160;3a and 3b)</li>
	<li>Record first nine coordinates using command+M keys (control+M for PCs).</li>
	<li>Advance frames until hyoid bone has reached maximum position in anterior and superior directions. Confirm maximum by advancing frames to insure descent of the hyoid begins on the following frame.</li>
	<li>Relocate points 1 - 5 to their new positions. These new positions will be recorded as coordinates 10 - 14.</li>
	<li>Relocate Point 9, which in turn becomes coordinate 18. Note: frames will likely vary for the next two steps.</li>
	<li>Locate frame depicting maximum laryngeal elevation. Adjust points 7 and 8, which will serve as coordinates 16 and 17.</li>
	<li>Find frame(s) representing the maximum excursion of UES, point 6 (coordinate 15). From maximum hyoid frame, locate frame where the bolus is impeded by the UES in the hypopharynx. Adjust coordinate point for UES from minimum frame to represent UES maximum coordinate 18.</li>
	<li>Record second nine coordinates using command+M keys (control+M for PCs).</li>
	<li>For coordinates 19 and 20 mark the edges of the scalar (a penny or 1.9 cm ring) at the axis representing the longest diameter of the radiopaque marker.</li>
	<li>Record scalar coordinates using command+M keys (control+M for PCs).</li>
</ol>
6. Transform coordinate data into kinematic measurements of interest using a macro enabled Excel file (the instructions for this macro are included on the file)
Note: Trigonometric calculations imbedded in the macros calculate kinematic measurements (Figure 4).
<ol>
	<li>Download &#34;CoordinateMapping.xlsm&#34; from the JoVE article page.</li>
	<li>Follow instructions on the spreadsheet to initialize the file. The initialization macro will create three sheets including: results, data, and input sheet. Note: Macintosh users must enable developer tools in Excel to run macros.</li>
	<li>Copy coordinates from the ImageJ results window and paste into the designated cell in the &#34;input sheet&#34;. Run the &#34;datacaptureline&#34; macro.</li>
	<li>Results will appear on the &#34;results&#34; sheet. Lines of coordinate data will appear on the &#34;data&#34; sheet.</li>
</ol>

<ol>
	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

The authors declare that they have no competing financial interests.

This study demonstrates the usefulness of a method using coordinate data of anatomical landmarks to calculate multiple kinematic measurements of hyolaryngeal movement in swallowing. Inter-rater reliability of six raters, including two novice raters, for coordinates and calculated variables was strong (ICC &#62; 0.90). Representative results from a random sample of healthy non-dysphagic adults showed differences in several kinematic variables in response to two bolus types. We also found that using different axes of refer...

The pharyngeal phase of swallowing is a complex process involving over twenty muscles and multiple skeletal elements to transfer a bolus from the oral cavity into the esophagus while protecting the airway. Preceding pharyngeal constriction, elements of the hyolaryngeal complex (hyoid bone, larynx, and associated structures including the upper esophageal sphincter) are displaced to convert a respiratory conduit into a digestive tract. The larynx is re-located anteriorly away from the trajectory of an oncoming bolus, and the upper esophageal sphincter is stretched open. Kinematic measurements taken from video fluoroscopic swallowing studies (also known as an MBS or Modified Barium Swallow Studies) are the primary research methodology for quantifying the multiple movements of the hyolaryngeal complex 1.
While quantitative video fluoroscopic measurements are useful for measuring swallowing function, different axes of reference and scalars of measurement result in findings that are incompatible among the various methods of kinematic measurements 2. The movement of the patient and fluoroscope under manual clinician control also confounds the accuracy of measuring this complex physiological process. More importantly, kinematic measurements do not necessarily reflect structure-to-function relationships important for evaluating disordered swallowing. Kinematics of the hyoid in particular have been designed to track movement in an anterior or superior direction in reference to an anatomical plane aligned with the vertebrae. However, this configuration does not represent the line of action of muscles that suspend the hyoid.
A two-sling mechanism of hyolaryngeal elevation in the pharyngeal phase of swallowing has been identified (Figure 1) 3,4. The suprahyoid muscles comprise the anterior muscular sling, and the long pharyngeal muscles comprise the posterior muscular sling. These muscles elevate various elements of the hyolaryngeal complex including the hyoid, larynx, and structures that form the upper esophageal sphincter.
Coordinate mapping of hyolaryngeal mechanics utilizes nine easily identifiable anatomical landmarks to map three skeletal levers and features of the hyolaryngeal complex representing attachment points of the anterior and posterior muscular slings (Figure 2). During swallowing, each skeletal lever and feature of the hyolaryngeal complex is in motion. By collecting coordinates, the system can be captured in any time frame. Trigonometric conversion of coordinates can be used to generate multiple kinematic measurements of hyolaryngeal movement during swallowing. Variables can be calculated for comparison with findings reported in the literature, or used to generate new measurements representing structure-to-function relationships of interest.
The primary aim of this paper is to demonstrate a method of generating multiple kinematic measurements calculated from a single set of anatomical landmark coordinates collected from Modified Barium Swallow (MBS) studies. We document the reliability of this method by using Intraclass correlation coefficients to determine the inter-rater reliability of 6 different investigators including one expert, three raters with experience, and two novices. From the kinematic results, differences in swallowing mechanics by bolus consistency are evaluated. Finally, the question proposed by Molefenter and Steele concerning the importance of the axis of reference used in measuring hyoid movement is addressed. To approach this question we compare measurements of hyoid excursion in reference to the vertebrae and in reference to the mandible, calculated from the same set of coordinates for both bolus types. If these two methods of measuring hyoid movement represent the same structure to function relationship, then the results should be strongly correlated.
For this study, 40 lateral view MBS studies were randomly selected from a collection of 139 normal studies under a research protocol approved by the Georgia Regents University Institutional Review Board and in research collaboration with the Evelyn Trammell Institute for Voice and Swallowing at the Medical University of South Carolina. To demonstrate the utility of this method, ten variables characterizing hyolaryngeal kinematics were calculated from the same set of coordinate data (Table 1). Seven of these calculated measurements have previously been used in the literature including: anterior and superior hyoid distance measurements in reference to the vertebrae 5; anterior and superior hyoid displacement as a ratio of C2-4 length, also in reference to the vertebrae 6; superior movement of the larynx in reference to the vertebrae 7; hyolaryngeal approximation 1; and maximum hyoid excursion in reference to the vertebrae 1. In addition, three novel measurements were calculated: pharyngeal shortening approximating the attachments of the palatopharyngeus muscle, laryngeal elevation following a line of action representing the stylopharyngeus, and hyoid excursion approximating the attachments of the suprahyoid muscles 4,8.
An expert head and neck anatomist (WP), three investigators with limited experience taking measurements (CJ, SR, TT), and two novice investigators (RS, JT) obtained coordinate mapping data using the protocol described below. The expert (WP) trained the three raters with experience, and these in turn trained the two novice raters. Inter-rater reliability of coordinate data and results calculated from coordinates by subject was determined by Intraclass correlation coefficients 9. Two tailed t-tests were performed on each variable to determine statistically significant differences in bolus types. A Pearson correlation coefficient and a coefficient of determination were used to evaluate agreement between results of hyoid excursion calculated with the vertebrae as an axis of reference versus hyoid excursion with the mandible as an axis of reference for the 5 ml thin-liquid swallow and 5 ml pudding swallow.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:339px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;&#160;</td>
			<td style="width:71px;">NIH</td>
			<td style="width:339px;">http://rsbweb.nih.gov/ij/download.html</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MacX Video Converter Free Edition (Mac)</td>
			<td style="width:71px;">Digiarty</td>
			<td style="width:339px;">&#160;http://www.macxdvd.com/mac-video-converter-free/</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickTime&#160;</td>
			<td style="width:71px;">Apple</td>
			<td style="width:339px;">http://support.apple.com/downloads/#QuickTime</td>
			<td>For Macintosh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;&#160;</td>
			<td style="width:71px;">NIH</td>
			<td style="width:339px;">http://rsbweb.nih.gov/ij/download.html</td>
			<td>For a PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPEG Streamclip (PC)&#160;</td>
			<td style="width:71px;">Squared 5</td>
			<td style="width:339px;">http://www.squared5.com</td>
			<td>For a PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickTime</td>
			<td style="width:71px;">Apple</td>
			<td style="width:339px;">http://support.apple.com/downloads/#QuickTime</td>
			<td>For a PC</td>
		</tr>
	</tbody>
</table>

coordinate mapping of hyolaryngeal mechanics in swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Intraclass correlation coefficients (ICC) of coordinates collected by six investigators who independently analyzed 80 video fluoroscopic files (two bolus trial from 40 subjects) ranged from ICC = 0.90 - 0.97. A breakdown of ICC&#39;s of coordinates by group is as follows: coordinates #1 - 5 (skeletal elements at minimum hyolaryngeal excursion) mean = 0.93, 0.91 - 0.95, 95% CI; coordinates #6 - 9 (hyolaryngeal complex at minimum hyolaryngeal excursion) mean = 0.94, 092 - 0.96, 95% CI; coordinates #10 - 14 (skeletal elemen...

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Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

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17.2K Views.  Georgia Regents University. Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

Video: Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...