The authors would like to thank past and current members of the Delaware Tendon Research Group who have assisted in data collection using this protocol. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Numbers R01AR072034, R01AR078898, F31AR081663, R21AR067390, and by Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD007490. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Research reported in this publication was also supported by grants provided by the Swedish Research Council for Sport Science, the Stockholm County Council (ALF project), and the Swedish Research Council. This work was&#160;also supported in part by a Promotion of Doctoral Studies Scholarship from the Foundation for&#160;Physical Therapy Research and by the Rheumatology Research Foundation Medical and Graduate Student Preceptorship.

The following protocol follows the guidelines set forth by and has been approved by the University Institutional Review Board to ensure safe and ethical human subject research. All subjects provided informed consent for participation in the research studies and dissemination of data. The full protocol takes approximately 20 minutes to perform by a trained sonographer. However, individual measurements may be taken per assessment need.
1. Marking of the skin
<ol>
	<li>Ask the participant to be seated at the end of a plinth with their legs hanging off the table.</li>
	<li>Palpate and mark the medial tibial plateau and most distal aspect of the medial malleolus bilaterally. Measure the distance between the ipsilateral markings to determine tibial length.</li>
	<li>Calculate 25% of the tibial length. Measure and mark this distance, distal from the medial tibial plateau, to indicate the location for gastrocnemius measurements (Figure 1).</li>
	<li>Calculate 30% of the tibial length. Measure and mark this distance, proximal from the most distal aspect of the medial malleolus, to indicate the location for soleus measurements (Figure 1).</li>
	<li>Record the 25% and 30% tibial length for longitudinally measuring the same location during future evaluations.</li>
	<li>Ask the participant to move into a prone position on the plinth with the knees fully extended and their feet hanging relaxed off the edge of the plinth with ankles in a resting position (Figure 2).</li>
	<li>Transfer the gastrocnemius and soleus markings to the posterior aspect of the lower leg. Palpate and mark the midpoint of the gastrocnemius (the distal most point between the two muscle bellies). Ask the participant to gently plantarflex against manual resistance to palpate this location.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65798/65798fig01.jpg" /> 
Figure 1: Skin measurement markings. Tibial length is measured as the distance from the medial tibial plateau to the most distal aspect of the medial malleolus. Soleus muscle thickness is measured at 30% of the tibial length proximal to the most distal aspect of the medial malleolus (blue mark). Gastrocnemius muscle CSA is measured at 25% of the tibial length distal to the medial tibial plateau (red mark). <a href="https://www.jove.com/files/ftp_upload/65798/65798fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65798/65798fig02.jpg" /> 
Figure 2: Participant positioning for ultrasound imaging. The participant is prone with knees fully extended and feet hanging relaxed in a resting ankle position off the edge of the plinth. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Ultrasound imaging
NOTE: All images will be taken on both legs. All images should be taken along the midline of the Achilles tendon. Use palpation of the medial and lateral borders of the Achilles tendon to determine midline location.
<ol>
	<li>Use B-mode ultrasound with a 5 cm linear array transducer with parameters set for visualization of the triceps surae. Set frequency to 10 MHz and depth of measurement to 3.5 cm for visualization. Adjust the foci between 0.75 cm and 1.75 cm with gain adjusted to 49 for optimal visualization of tissue. 
	NOTE: Depth, foci, and gain may be adjusted as appropriate for visualization.</li>
	<li>Place ultrasound gel on the posterior calcaneus. Holding the probe on the long axis, visualize the proximal calcaneal notch and align the notch with the midpoint of the ultrasound probe. Mark this location on the skin (Figure 3).</li>
	<li>Marking the skin for measurement of Achilles tendon thickness and CSA will vary depending on the participant. Use the following guidelines to determine this position.
	<ol>
		<li>For healthy individuals, measure 2 cm proximal from the proximal calcaneal notch and mark this location on the skin.</li>
		<li>For individuals with Achilles tendinopathy, visualize the thickest portion of the Achilles tendon with the ultrasound probe on the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch forlongitudinally measuring the same location during future evaluations.</li>
		<li>For individuals with Achilles tendinopathy without visible fusiform thickening, mark the skin at the location of most tendon pain upon palpation.&#160;Record the distance of this marking from the&#160;proximal calcaneal notch for longitudinally measuring the same location during future evaluations.</li>
		<li>For individuals post Achilles tendon rupture, visualize the location of rupture with the ultrasound probe in the long axis and mark this location on the skin. Record the distance of this marking from the proximal calcaneal notch for longitudinally measuring the same location during future evaluations.</li>
		<li>For the uninvolved limb, make the skin marking at the same distance from the proximal calcaneal notch as the involved limb.</li>
	</ol>
	</li>
	<li>Gastrocnemius tendon length (Full tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the gastrocnemius midpoint until the myotendinous junction is visualized. This will end the image. Take three images of the Achilles&#39; full tendon length (Figure 4).</li>
	<li>Soleus tendon length (Free tendon length): Using an extended field-of-view and holding the probe on the long axis, begin the image at the calcaneus by visualizing the Achilles tendon insertion. Glide the probe proximally along the midline of the Achilles tendon towards the mark at the soleus until the soleus myotendinous junction is visualized. Take three images of the Achilles&#39; free tendon length.</li>
	<li>Achilles tendon thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Take three images of Achilles tendon thickness.</li>
	<li>Achilles tendon cross-sectional area: Using the B-mode view, place a gel standoff pad on the skin marking made for Achilles tendon thickness and CSA (step 2.3). Holding the probe on the short axis, take three images of the Achilles tendon cross-section. 
	NOTE: Tendon is of an anisotropic quality; depending on the angle of the sound waves, it will reflect back, making it look bright, or reflect away, making it look dark (Figure 5). Therefore, toggling the probe back and forth will help with visualizing the border of the tendon.</li>
	<li>Soleus thickness: Using the B-mode view, place the probe on the long axis on the skin marking made for the soleus (30% of the tibial length from the distal aspect of the medial malleolus). Visualize the anterior border of the soleus muscle. Take three images for soleus thickness19.
	<ol>
		<li>To distinguish between the soleus and flexor hallucis longus, have the participant actively flex and extend the great toe. This will cause movement of the fibers of the flexor hallucis longus without moving the soleus fibers.</li>
	</ol>
	</li>
	<li>Gastrocnemius cross-sectional area: Using an extended field-of-view, hold the ultrasound probe on the short axis and in line with the mark for the gastrocnemius (25% of the tibial length from the medial tibial plateau). Visualize the medial border of the medial gastrocnemius and begin the image by gliding the probe from medial to lateral until the lateral border of the lateral gastrocnemius is visualized. Take three images for gastrocnemius CSA13.
	<ol>
		<li>To capture the most medial aspect of the medial gastrocnemius it may be needed to&#160;ask the participant to abduct their legs and internally rotate the hip slightly. If this is required, ensure the participant is not plantarflexing their foot, as this will impact measurements.</li>
	</ol>
	</li>
	<li>Using the B-mode view, determine the presence or absence of calcifications within the Achilles tendon (Figure 6D) and/or at the Achilles tendon insertion (enthesis; Figure 6C). These calcifications are unattached from the calcaneus and appear as hyperechoic areas with a shadow below, present in multiple planes of view. Take an image to confirm the findings. Note whether the calcifications occur within the Achilles tendon or at the enthesis.</li>
	<li>Using the B-mode view, determine the presence or absence of bursitis. This is visualized by a hypoechoic area deep to the Achilles tendon at the proximal calcaneus (retrocalcaneal bursitis) and/or superficial to the Achilles tendon insertion (subcutaneous calcaneal bursitis). Take an image to confirm the findings (Figure 6B).</li>
	<li>Using the Doppler setting on the ultrasound, assess for the presence or absence of neovascularization within the Achilles tendon.
	<ol>
		<li>Position the Doppler box (i.e., region of interest) on the tendon, hold the ultrasound probe still, and avoid pressing down or distending the tissue as this may occlude and prevent visualization of blood flow.</li>
		<li>Scan the length of the Achilles free tendon, being sure to assess each portion of the tendon without movement of the probe to prevent artifact. If there is vasculature visible within or in contact with the tendon, take three, 3 s videos in the region with maximal blood flow (Figure 6A).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65798/65798fig03.jpg" /> 
Figure 3: Achilles tendon thickness and CSA measurement locations. Extended field of view ultrasound images of (A) a healthy and (B) tendinopathic Achilles tendon. Dotted lines indicate tendon borders. Red arrows indicate measurement locations. Yellow lines indicate the distance proximal to the proximal calcaneal notch (white arrow). In the case of thickening at the 2 cm mark (B), healthy tendon thickness should be taken at a healthy area in the free tendon (blue arrow). <a href="https://www.jove.com/files/ftp_upload/65798/65798fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65798/65798fig04.jpg" /> 
Figure 4: Achilles tendon length measurements. Extended field of view ultrasound image of the Achilles tendon. Free tendon length (yellow line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the soleus myotendinous junction (MTJ). Full tendon length (red line) is measured from the proximal Achilles tendon insertion on the proximal calcaneal notch to the gastrocnemius myotendinous junction (MTJ)10. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65798/65798fig05.jpg" /> 
Figure 5: Tendon anisotropy. Achilles tendon anisotropy occurs when the ultrasound waves are not perpendicular to the structure. Tilting the ultrasound probe will cause the tendon structure to appear (A) hyperechoic or (B) hypoechoic depending on the relationship of ultrasound waves to the tendon. Dotted lines indicate tendon borders. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65798/65798fig06.jpg" /> 
Figure 6: Additional ultrasound imaging findings. (A) Neovascularization, (B) retrocalcaneal bursitis, (C) entheseal calcifications, (D) intratendinous calcifications. Areas colored red and yellow indicate blood flow within the tendon region of interest (yellow box). Dotted lines indicate bursa borders. Arrows indicate calcifications20. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Ultrasound measurements
NOTE: Ultrasound measurements can be made on the machine based on the settings and tools for the respective ultrasound machine. Images can also be exported for taking measurements on software such as Osirix DICOM viewer. The average of the three trials for each measure will be used for analysis.
<ol>
	<li>Gastrocnemius tendon length (full tendon length): Open the longer extended field-of-view tendon image (taken in step 2.4). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the gastrocnemius myotendinous junction (Figure 4).</li>
	<li>Soleus tendon length (free tendon length): Open the shorter extended field-of-view tendon image (taken in step 2.5). Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to the soleus myotendinous junction (Figure 4).</li>
	<li>Tendon thickness at 2 cm: Open the shoter extended field-of-view tendon image. Measure from the most proximal point of the tendon insertion (proximal calcaneal notch) to 2 cm, with the end of the measurement on the deep border of the tendon. Measure from this point on the deep border of the tendon to the direct superficial aspect of the tendon to get the 2 cm tendon thickness. This measure should represent healthy tendon thickness (Figure 7A).
	<ol>
		<li>In the case of Achilles tendinopathy, if the tendon is thickened at this 2 cm distance, locate a healthy area in the free tendon to take this measurement and document the distance from the proximal calcaneal notch (Figure 7B). 
		NOTE: Thickness is used to describe the raw thickness of the tendon from the most superficial to deep.</li>
	</ol>
	</li>
	<li>Tendon cross-sectional area: Open image taken in step 2.7.&#160;Visually identify the borders of the Achilles tendon and outline the circumference of the tendon (Figure 8A).</li>
	<li>Tendon thickness: Open image taken in step 2.6.&#160;At the center of the image, measure the Achilles tendon from the superficial border to the deep border of the tendon (Figure 8B).</li>
	<li>Soleus thickness: Open image taken in step 2.8. At the center of the image, measure from the superficial border to the deep border of the soleus muscle (Figure 9).</li>
	<li>Gastrocnemius cross-sectional area: Open image taken in step 2.9.&#160;Visualize the borders of both the medial and lateral heads of the gastrocnemius and outline the medial gastrocnemius and then the lateral gastrocnemius heads (Figure 10).</li>
	<li>Achilles tendon thickening: Use this calculation to determine the degree of thickening of the tendon in Achilles tendinopathy. Subtract thickness at 2 cm (or adjusted location; Figure 7A) from tendon thickness (Figure 7B) to obtain tendon thickening (measurement in step 3.5 minus measurement in step 3.3).</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65798/65798fig07.jpg" /> 
Figure 7: Achilles tendon thickening. Achilles tendon thickening is calculated by subtracting (A) the thickness of the healthy portion of the tendon from (B) the thickness of the thickest portion of the tendon31. Dotted lines indicate tendon borders. Red lines indicate tendon thickness. Yellow lines indicated a 2 cm distance proximal to the proximal calcaneal notch. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65798/65798fig08.jpg" /> 
Figure 8: Achilles tendon cross-sectional area and thickness. Ultrasound images of the Achilles tendon in (A) cross-section and (B) longitudinal view at the thickest portion. Dotted lines indicate tendon borders. The red line indicates tendon thickness. A cross-section of the tendon is shown with and without a border for clarity. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65798/65798fig09.jpg" /> 
Figure 9: Soleus muscle thickness. Ultrasound image of the soleus muscle. Dotted lines indicate muscle borders. The red line indicates muscle thickness. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65798/65798fig10.jpg" /> 
Figure 10: Gastrocnemius muscle cross-sectional area. Extended field of view ultrasound image of medial and lateral gastrocnemius muscles in cross-section. Dotted lines indicate tendon borders. <a href="https://www.jove.com/files/ftp_upload/65798/65798fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Ultrasound Evaluation of Achilles Tendon Morphology

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T., Stengaard-Pedersen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+in+evaluation+of+Achilles+and+patella+tendon+thickness.">Ultrasonography in evaluation of Achilles and patella tendon thickness.</a> Ultraschall Med. 29 (1), 60-65 (2007).</li><li>Dudley-Javoroski, S., McMullen, T., Borgwardt, M. R., Peranich, L. M., Shields, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+responsiveness+of+musculoskeletal+ultrasound+in+subjects+with+and+without+spinal+cord+injury.">Reliability and responsiveness of musculoskeletal ultrasound in subjects with and without spinal cord injury.</a> Ultrasound Med Biol. 36 (10), 1594-1607 (2010).</li><li>Zellers, J. A., Cortes, D. H., Pohlig, R. T., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+morphology+and+mechanical+properties+assessed+by+ultrasound+show+change+early+in+recovery+and+potential+prognostic+ability+for+6-month+outcomes.">Tendon morphology and mechanical properties assessed by ultrasound show change early in recovery and potential prognostic ability for 6-month outcomes.</a> Knee Surg Sports Traumatol Arthrosc. 27 (9), 2831-2839 (2019).</li><li>Zellers, J. A., Bley, B. C., Pohlig, R. T., Alghamdi, N. H., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+pathology+on+diagnostic+ultrasound+and+relationship+to+patient+demographics+in+individuals+with+insertional+Achilles+tendinopathy.">Frequency of pathology on diagnostic ultrasound and relationship to patient demographics in individuals with insertional Achilles tendinopathy.</a> Int J Sports Phys Ther. 14 (5), 761-769 (2019).</li><li>de Jonge, S., 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=Incidence+of+midportion+Achilles+tendinopathy+in+the+general+population.">Incidence of midportion Achilles tendinopathy in the general population.</a> Br J Sports Med. 45 (13), 1026-1028 (2011).</li><li>Lemme, N. J., Li, N. Y., DeFroda, S. F., Kleiner, J., Owens, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Achilles+tendon+ruptures+in+the+United+States:+athletic+and+nonathletic+injuries+from+2012+to+2016.">Epidemiology of Achilles tendon ruptures in the United States: athletic and nonathletic injuries from 2012 to 2016.</a> Orthop J Sports Med. 6 (11), 2325967118808238 (2018).</li><li>Jackson, J. B., Chu, C. H., Williams, K. A., Bornemann, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+ultrasonographic+parameters+of+the+posterior+tibial,+peroneal,+and+Achilles+tendons.">Normal ultrasonographic parameters of the posterior tibial, peroneal, and Achilles tendons.</a> Foot Ankle Spec. 12 (5), 480-485 (2019).</li><li>Schmidt, W. A., Schmidt, H., Schicke, B., Gromnica-Ihle, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+reference+values+for+musculoskeletal+ultrasonography.">Standard reference values for musculoskeletal ultrasonography.</a> Ann Rheum Dis. 63 (8), 988-994 (2004).</li><li>Ackermann, P. W., Hart, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Influences on Risk for Tendon Disorders. , (2016).</li><li>Westh, E., 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=Effect+of+habitual+exercise+on+the+structural+and+mechanical+properties+of+human+tendon,+in+vivo,+in+men+and+women.">Effect of habitual exercise on the structural and mechanical properties of human tendon, in vivo, in men and women.</a> Scand J Med Sci Sports. 18 (1), 23-30 (2008).</li><li>Cassel, 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=Prevalence+of+Achilles+and+patellar+tendinopathy+and+their+association+to+intratendinous+changes+in+adolescent+athletes.">Prevalence of Achilles and patellar tendinopathy and their association to intratendinous changes in adolescent athletes.</a> Scand J Med Sci Sports. 25 (3), e310-e318 (2015).</li><li>Hirschm&#252;ller, 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=Achilles+tendon+power+Doppler+sonography+in+953+long+distance+runners+-+a+cross+sectional+study.">Achilles tendon power Doppler sonography in 953 long distance runners - a cross sectional study.</a> Ultraschall Med. 31 (4), 387-393 (2010).</li><li>Radovanovi&#263;, G., Bohm, S., Arampatzis, A., Legerlotz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Achilles+tendinopathy+the+symptomatic+tendon+differs+from+the+asymptomatic+tendon+while+exercise+therapy+has+little+effect+on+asymmetries-an+ancillary+analysis+of+data+from+a+controlled+clinical+trial.">In Achilles tendinopathy the symptomatic tendon differs from the asymptomatic tendon while exercise therapy has little effect on asymmetries-an ancillary analysis of data from a controlled clinical trial.</a> J Clin Med. 12 (3), 1102 (2023).</li><li>Corrigan, P., Cortes, D. H., Pontiggia, L., Silbernagel, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+degree+of+tendinosis+is+related+to+symptom+severity+and+physical+activity+levels+in+patients+with+midportion+Achilles+tendinopathy.">The degree of tendinosis is related to symptom severity and physical activity levels in patients with midportion Achilles tendinopathy.</a> Int J Sports Phys Ther. 13 (2), 196-207 (2018).</li><li>Enriquez, J. L., Wu, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+ultrasound+equipment+and+knobology.">An introduction to ultrasound equipment and knobology.</a> Crit Care Clin. 30 (1), 25-45 (2014).</li><li>Jacobson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of Musculoskeletal Ultrasound 3rd Edition. , (2018).</li><li>Scanlan, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonographic+artifacts+and+their+origins.">Sonographic artifacts and their origins.</a> AJR Am J Roentgenol. 156 (6), 1267-1272 (1991).</li><li>Gimber, L. H., 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=Artifacts+at+musculoskeletal+US:+resident+and+fellow+education+feature.">Artifacts at musculoskeletal US: resident and fellow education feature.</a> Radiographics. 36 (2), 479-480 (2016).</li></ol>

The authors have no conflicts of interest to disclose.

Critical steps in the protocol to ensure the validity and reliability of the methods include the use of skin markings on participants to guide the ultrasound exam and the required training of individuals performing the measurements used in research studies. Skin markings at specific documented locations assist in the ability to assess variables of interest consistently and accurately at the same locations in the same individual over time. Furthermore, applying skin markings to different individuals in the same systematic...

The Achilles tendon originates at the myotendinous junctions of the gastrocnemius and soleus muscles and inserts on the posterior calcaneus. The Achilles tendon consists primarily of densely packed, organized collagen tissue arranged in a hierarchical fashion to maximize tensile strength1. Despite its ability to withstand heavy forces, the Achilles tendon is susceptible to several types of injuries throughout one&#39;s lifespan. These injuries, such as Achilles tendinopathy and Achilles tendon ruptures, are often accompanied by changes in the structure of the triceps surae and surrounding tissues. In Achilles tendinopathy, patients often exhibit fusiform tendon thickening, tendinosis, collagen disorganization, and neovascularization, a process of proliferation of vascular and neural tissue into the tendon2. Additionally, pathological changes associated with Achilles tendinopathy include paratendinitis, intratendinous, and/or entheseal calcifications, and bursitis2,3. After Achilles tendon ruptures, structural changes are common occurrences, irrespective of treatment, and include Achilles tendon thickening and increased tendon length4,5. Furthermore, muscular changes, such as triceps surae muscular atrophy, are also commonly associated with Achilles tendon injuries5,6.
The ability to assess triceps surae and surrounding tissue structures provides valuable insight into structural integrity, tissue quality, and size, which are known to relate to symptoms, function, and prognosis4,7,8,9. Ultrasound imaging is a reliable and valid assessment tool of these structures, including, but not limited to, Achilles tendon length10, thickness10,11, cross-sectional area (CSA)12, gastrocnemius anatomical CSA13, and neovascularization14,15. Assessment of these measures provides valuable insight into the understanding of healthy triceps surae tissue, as well as quantification of structural alterations to evaluate injury risk, severity, and recovery, as well as understanding healthy tissue qualities16.
Despite the clinical and research utility of ultrasound imaging in assessing triceps surae structure, there are often differences in imaging techniques and measurement parameters among clinical and research studies17,18. As a result, comparisons between studies are difficult. Therefore, the purpose of this methods paper is to describe a valid and reliable protocol for efficiently evaluating triceps surae muscle and tendon structure using musculoskeletal ultrasound imaging. This protocol aims to demonstrate the feasibility of incorporating this tool into research and clinical settings through its entirety or as specific parts in healthy and injured individuals. In addition, representative values for healthy and injured triceps surae are provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquaflex Stand Off Pad</td>
			<td>Parker Laboratories&#160;</td>
			<td>E8317C</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasonic ultrasound Gel</td>
			<td>Parker Laboratories&#160;</td>
			<td>E8365AF</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Array Ultrasound Probe L4-12t-RS</td>
			<td>GE Healthcare</td>
			<td>5495987</td>
			<td></td>
		</tr>
		<tr>
			<td>LOGIC e Ultrasound</td>
			<td>GE Healthcare</td>
			<td>E8349PA</td>
			<td></td>
		</tr>
		<tr>
			<td>Osirix Dicom Viewer</td>
			<td>Pixmeo SARL</td>
			<td></td>
			<td>Software for measurements</td>
		</tr>
	</tbody>
</table>

measurement of healthy and injured triceps surae morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally classified as an overuse injury associated with fusiform tendon thickening, neovascularization, and interstitial tendon degeneration. Current literature suggests these structural changes are associated with&#160;symptoms and lower physical activity levels, as well as&#160;symptoms and lower extremity function in the long term. Surgically and non-surgically managed Achilles tendon ruptures result in increased tendon cross-sectional area (CSA) and a lengthened Achilles tendon. Both structural outcomes have clinical implications, as larger CSA positively predicts function, whereas increased tendon lengthening predicts reduced function after Achilles tendon rupture. Given the relationship between structural changes associated with Achilles tendon injuries for both injury severity and injury recovery, it is critical to be able to quantify Achilles tendon structure reliably and accurately. Silbernagel&#39;s group has established a valid and reliable method for efficiently evaluating triceps surae muscle and tendon structure. In this protocol, B-mode musculoskeletal ultrasound imaging is used to measure triceps surae structure, including Achilles tendon thickness and CSA, soleus thickness, and the presence of additional findings (calcifications and bursitis). B-mode extended field-of-view is used to measure Achilles tendon length and gastrocnemius anatomical CSA. Finally, power Doppler is used to identify intratendinous neovascularization. Quantification of triceps surae structure allows for comparison between limbs as well as longitudinal changes in response to exercise and treatment for healthy individuals and those with Achilles tendon injuries. This protocol has been used in many research studies to date and proves valuable in understanding the relationship between tendon structure and injury development, severity, and recovery. As ultrasound devices are becoming more affordable and portable, this protocol proves promising as a clinical tool, given its quick and efficient methods.

The measures in this protocol have been demonstrated to be reliable and valid10,20. Excellent reliability has been reported for extended field-of-view Achilles tendon length to gastrocnemius (interclass correlation coefficient (ICC): 0.944) and Achilles tendon length to soleus (ICC: 0.898)10. The minimal detectable change at the group level (MDCgroup) for Achilles tendon length measures is 0.43 cm for length to gastrocnemius and...

Watch this Scientific Journal Video about Measurement of Healthy and Injured Triceps Surae Morphology at JoVE.com

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Measurement of Healthy and Injured Triceps Surae Morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally ...

measurement-of-healthy-and-injured-triceps-surae-morphology

Research

JoVE Journal

Medicine

573 Views.  University of Delaware. Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons. 

Video: Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries — Research and Future Directions

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Experimental Protocol of a Three-minute, All-out Arm Crank Exercise Test in Spinal-cord Injured and Able-bodied Individuals

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be small; therefore, sensitive tools are fundamental to exercise physiology. There are currently many exercise tests that allow for the examination of exercise capacity in able-bodied athletes, with protocols mainly for lower-body or whole-body exercise. There is a trend to test athletes in a sport-specific setting that closely resembles the actions that the participants are used to performing. Only a few protocols test short-term, high-intensity exercise capacity in participants with an impairment of the lower body. Most of these protocols are very sport-specific and are not applicable to a wide range of athletes. One well-known test protocol is the 30 s Wingate test, which is well-established in cycling and in arm crank exercise testing. This test analyzes high-intensity exercise performance over a 30 s time duration. In order to monitor exercise performance over a longer duration, a different method was modified for application to the upper body. The 3 min, all-out arm crank ergometer test allows athletes to be tested in a manner specific to 1,500 m wheelchair racing (in terms of exercise duration), as well as to upper body exercises such as rowing or hand-cycling. In order to increase the reliability with identical test conditions, it is crucial to precisely replicate settings such as the resistance (i.e., torque factor) and the position of the participants (i.e., the height of the crank, the distance between the crank and the participant, and the fixation of the participant). Another important issue concerns the beginning of the exercise test. Fixed revolutions per minute are required to standardize the test conditions for the start of the exercise test. This exercise protocol shows the importance of accurate operations to reproduce identical test conditions and settings.

We present a protocol to test the aerobic and anaerobic power of the upper body muscles over a duration of 3 min in able-bodied as well as in paraplegic and tetraplegic individuals. The protocol presents specific modifications in its application for upper-body exercise in individuals with or without disability.

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Virtual Prism Adaptation Therapy: Protocol for Validation in Healthy Adults

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is imperative for the successful management of hemispatial neglect. However, the clinical use of various interventions is limited in real clinical practice. Prism adaptation therapy is one of the most evidence-based rehabilitation modalities to treat hemispatial neglect. To overcome any possible shortcoming that may occur with prism therapy, we developed a new system using immersive virtual reality and depth-sensing camera to create a virtual prism adaptation therapy (VPAT). To validate the VPAT system, we designed an experimental protocol investigating the behavioral errors and changes in cortical activation via the VPAT system. Cortical activation was measured by functional near infrared spectroscopy (fNIRS). The experiment consisted of four phases. All four included clicking, pointing or rest applied to right-handed healthy people. Clicking versus pointing was used for investigating the cortical region related with the gross motor task, and pointing with VPAT versus pointing without VPAT was used for investigating the cortical region associated with visuospatial perception. The preliminary results from four healthy participants showed that pointing errors by the VPAT system was similar to the conventional prism adaptation therapy. Further analysis with more participants and fNIRS data, as well as a study in patients with stroke may be required.

This experimental protocol demonstrates the use of virtual prism adaptation therapy (VPAT) in healthy adults and the association between VPAT and functional near infrared spectroscopy to determine the effect of VPAT on cortical activation. Results suggest that VPAT may be feasible and could induce similar behavioral adaptation as conventional prism adaptation therapy.

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is ...