Name: obtaining quality extended field-of-view ultrasound images of skeletal muscle to measure muscle fascicle length
Uploaded: 2020-12-14T14:00:00
Description: Watch this Scientific Journal Video about Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length at JoVE.com

We would like to thank Vikram Darbhe and Patrick Franks for their experimental guidance. This work is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1324585 as well as NIH R01D084009 and F31AR076920. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation or NIH.

Northwestern University&#8217;s Institutional Review Board (IRB) approved the procedures of this study. All participants enrolled in this work gave informed consent prior to beginning the protocol detailed below. 
NOTE: The specific ultrasound system used in this study had EFOV-US capabilities and was adopted because we were able to review details about and validity assessments for the algorithm in the scientific literature22,26; multiple other systems with ...

Critical steps in the protocol.
There are a few critical components to obtaining quality EFOV-US images that yield valid and reliable fascicle length measures. First, as indicated in method 1.1.2 it is essential that the sonographer take time to become familiar with the anatomy of the muscle being imaged as well as surrounding muscles, bones, and other soft tissue structures. This will improve the sonographer&#8217;s ability to image the correct muscle and determine if multipl...

Fascicle length is an important parameter of skeletal muscle architecture, which overall is indicative of a muscle&#8217;s ability to produce force1,2. Specifically, a muscle&#8217;s fascicle length provides insight into the absolute range of lengths over which a muscle can generate active force3,4. For example, given two muscles with identical values for all isometric force-generating parameters (i.e., average sarcomere length, pennation angle, physiological cross sectional area, contraction state, etc.) except for fascicle length, the muscle with the longer fascicles would produce its peak force at a longer length and would produce force over a wider range of lengths than the muscle with shorter fascicles3. Quantification of muscle fascicle length is important for understanding both healthy muscle function and changes in a muscle&#8217;s force-generating capacity, which can occur as a result of altered muscle use (e.g., immobilization5,6, exercise intervention7,8,9, high heel wearing10) or a change in the muscle&#8217;s environment (e.g., tendon transfer surgery11, limb distraction12). Measurements of muscle fascicle length were originally obtained through ex vivo cadaveric experiments that allow for direct measurement of dissected fascicles13,14,15,16. The valuable information provided by these ex vivo experiments led to an interest in implementing in vivo methods17,18,19 to address questions that could not be answered in cadavers; in vivo methods allow for quantification of muscle parameters in a native state as well as at different joint postures, different muscle contraction states, different loading or unloading states, and across populations with differing conditions (i.e. healthy/injured, young/old, etc.). Most frequently, ultrasound is the method employed to obtain in vivo muscle fascicle lengths18,19,20; it is quicker, less expensive, and easier to implement than other imaging techniques, such as diffusion tensor imaging (DTI)18,21.
Extended field-of-view ultrasound (EFOV-US) has been demonstrated to be a valid and reliable method for measuring muscle fascicle length in vivo. While commonly implemented, traditional ultrasound (T-US) has a field-of-view which is limited by the ultrasound transducer&#8217;s array length (typically between 4 and 6 cm, although there are probes that extend to 10 cm10)18,20. To overcome this limitation, Weng et al. developed an EFOV-US technology that automatically acquires a composite, two-dimensional &#8220;panoramic&#8221; image (up to 60 cm long) from a dynamic, extended distance scan22. The image is created by fitting together, in real-time, a sequence of traditional, B-mode ultrasound images as the transducer dynamically scans the object of interest. Because sequential T-US images have large overlapping regions, the small differences from one image to the next can be used to calculate the probe motion without the use of external motion sensors. Once the probe motion between two consecutive images is calculated, the &#8220;current&#8221; image is merged successively with the preceding images. The EFOV-US method allows direct measurement of long, curved muscle fascicles and has been demonstrated to be reliable across muscles, trials, and sonographers23,24,25 and valid for both flat and curved surfaces23,26.
Implementing ultrasound to measure muscle fascicle length in vivo is not trivial. Unlike other imaging techniques that involve more automated protocols (i.e., MRI, CT), ultrasound is dependent on sonographer skill and anatomical knowledge27,28. There is concern that probe misalignment with the fascicle plane may cause substantial error in fascicle measures. One study demonstrates little difference (on average &#60; 3 mm) in measures of fascicle length taken using ultrasound and DTI MRI but also shows that measurement precision is low (standard deviation of difference ~12 mm)29. Still, it has been shown that a novice sonographer, with practice and guidance from an experienced sonographer, can obtain valid meaures using EFOV-US23. Thus, efforts should be made to demonstrate appropriate protocols to reduce human error and improve accuracy of measurements obtained using EFOV-US. Ultimately, developing and sharing appropriate protocols may expand the number of experimenters and laboratories that can reproduce fascicle length data from the literature or obtain novel data in muscles which have not yet been studied in vivo.
In this protocol, we demonstrate how to implement the EFOV-US method to obtain high quality musculoskeletal images that can be used to quantify muscle fascicle length. Specifically, we address (a) collecting EFOV-US images of a single upper limb and a single lower limb muscle (b) determining, in real-time, the &#8220;quality&#8221; of the EFOV-US image, and (c) quantifying muscle architecture parameters offline. We provide this detailed guide to encourage the adoption of the EFOV-US method for obtaining muscle fascicle length data in muscles that have gone unstudied in vivo due to their long fascicles.

<table>
	<tbody>
		<tr>
			<td>14L5 linear transducers</td>
			<td>Siemens</td>
			<td>10789396</td>
			<td></td>
		</tr>
		<tr>
			<td>Acuson S2000 Ultrasound System</td>
			<td>Siemens</td>
			<td>10032746</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable chair (Biodex System)</td>
			<td>Biodex Medical Systems</td>
			<td>System Pro 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin Marker Medium Tip</td>
			<td>SportSafe</td>
			<td>n/a</td>
			<td>Multi-color 4 Pack recommended</td>
		</tr>
		<tr>
			<td>Ultrasound Gel - Standard 8 Ounce Non-Sterile Fragrance Free Glacial Tint</td>
			<td>MediChoice, Owens &#38;Minor</td>
			<td>M500812</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining quality extended field-of-view ultrasound images of skeletal muscle to measure muscle fascicle length

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force generating capacity. However, over 90% of all upper limb muscles and 85% of all lower limb muscles have optimal fascicle lengths longer than the field-of-view of common traditional ultrasound (T-US) probes. A newer, less frequently adopted method called extended field-of-view ultrasound (EFOV-US) can enable direct measurement of fascicles longer than the field-of-view of a single T-US image. This method, which automatically fits together a sequence of T-US images from a dynamic scan, has been demonstrated to be valid and reliable for obtaining muscle fascicle lengths in vivo. Despite the numerous skeletal muscles with long fascicles and the validity of the EFOV-US method for making measurements of such fascicles, few published studies have utilized this method. In this study, we demonstrate both how to implement the EFOV-US method to obtain high quality musculoskeletal images and how to quantify fascicle lengths from those images. We expect that this demonstration will encourage the use of the EFOV-US method to increase the pool of muscles, both in healthy and impaired populations, for which we have in vivo muscle fascicle length data.

Extended field-of-view ultrasound (EFOV-US) was implemented to obtain images from the long head of the biceps brachii and the tibialis anterior in 4 healthy volunteers (Table 1). Figure 1 shows what EFOV-US images of both muscles imaged in this representative imaging session and highlights important aspects of each image such as muscle aponeurosis, central tendon, fascicle path, etc. After the imaging session was over, 3 qualitatively &#8220;good&#8221; images (<strong class...

Watch this Scientific Journal Video about Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length at JoVE.com

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force ...

Being able to obtain quality extended field of view ultrasound images can increase the number of muscles for which we have in vivo fascicle length data. The main advantage of extended field of view ultrasound is that it can enable direct measurement of longer curved muscle fascicles which cannot be captured with traditional ultrasound. This method may be applied to the over 90%of upper limb and 85%of lower limb muscles with optimal fascicle lengths longer than the field of view of common ultrasound probes.Successful implementation of this method requires a good understanding of musculoskeletal anatomy. Practice obtaining images and practice assessing the quality of images. Begin by taking time to become familiar with the systems method for obtaining EFOV-US and with the anatomy of the muscles of interest.After explaining the study protocol, seat the participant in an adjustable chair that can be locked in place, and adjust the chair so that the participant is as comfortable as possible while still providing access to the muscle of interest. Place the joint that the muscle of interest spans in a posture that can be controlled and repeated and use clinical guidance to locate anatomical landmarks. Using the ISB standards for defining the joint coordinate system, mark the anatomical landmarks with a skin-safe marker.Align the center of a handheld goniometer with the axis of rotation of the joint and align the arms of the goniometer with the joint's segments, then, measure the joint angle. Then use cloth straps to secure the limb to minimize movement during the imaging protocol. For image acquisition, plug in and turn on the ultrasound system and ensure that the exam is set to musculoskeletal, the transducer is set to the transducer in use, and the transmit frequency is set between five to 17 megahertz.In the system's software, set the foot switch to start-stop the imaging. If the foot switch in use has multiple pedals, set additional pedals to freeze or pause and to print or store the image. Next, apply a generous amount of ultrasound gel to the head of the transducer and place the transducer onto the participant's skin in the approximate region of interest.Move the transducer across the shorter axis plane of the muscle keeping the indicator pointed laterally. Identify the muscle of interest in the short axis plane perpendicular to the muscle fiber direction and move the transducer distally and proximally to acquire a full visualization of the muscle path using the skin-safe markers to mark important anatomical landmarks as they are identified. Once the location of the muscle has been identified and marked, move the ultrasound transducer in the long axis plane with the indicator pointed distally.When either the distal or proximal end of the muscle has been identified, rotate and tilt the transducer to identify the fascicle plane at that point and mark the skin when the correct transducer position has been established. Once the approximate fascicle plane has been established along the entire desired length to be scanned, practice following the muscle path and set the ultrasound system to the extended field of view mode. Starting at one end of the muscle, click the foot switch to start the image acquisition and slowly and continuously move the ultrasound transducer along the long axis.When the end of the muscle has been reached, click the foot switch to end image acquisition. To optimize image visibility and clarity, if the image acquisition ends before the desired length of the muscle can be captured, increase the depth of the image. To adjust the focus, place the focus arrow in the lower 1/2 of the image just below the muscle of interest.Ensure that the gain is balanced through the depth of the image and use the indicator to determine the optimal speed for moving the transducer. Once qualitatively good images have been collected, export the images as uncompressed DICOM images. Adjust the chair so that the participant is comfortable and the tibialis anterior can be accessed with ultrasound.Then, locate and mark anatomical landmarks and make joint angle measures. After finding the location of the fascicle plane and practicing capturing images, capture an EFOV-US image of the tibialis anterior. When all of the images have been acquired, open a long axis image of interest in an appropriate image analysis software program.Identify the connective tissues surrounding the muscle as bright hyperechoic lines indicating the muscle boundaries around a dark hypoechoic shape and check that the image does not have excessive non-anatomical bending, gaps in the image, or a jagged, flexible ruler line over the image. If the image is missing one or more of these tissue structures, deem the image qualitatively poor. To quantify the muscle fascicle length, first, open the image of interest in ImageJ and use the straight line tool to draw a 10 millimeter straight line on the ruler on the side of the ultrasound image.Select analyze and measure to measure the line. If the image properties have been preserved, the length of the straight line should be approximately one centimeter. To measure the fascicle lengths in the image, right click on the straight line tool to select segmented line.Click one end of the fascicle to be measured and continuously click along the muscle path to ensure that the curvature in the fascicle has been captured. Then, double-click at the end of the fascicle and select analyze and measure to measure the length of the line. In these representative analyses, extended field of view ultrasound was used to obtain images from the long head of the biceps brachii and the tibialis anterior in four healthy volunteers.Extended field of view ultrasound imaging of these muscles was used to observe important aspects of the tissues such as central tendon and fascicle path. After imaging, qualitatively good images were then analyzed for each muscle in each individual. ImageJ was implemented to measure four fascicles with paths that could be convincingly visualized from their origin to insertion and that were located in different portions of the muscle in each image.The average fascicle lengths obtained in this study for the biceps brachii and the tibialis anterior were within the range of previously reported fascicle lengths. As it can be difficult to determine which images are appropriate for fascicle evaluation, here, representative, qualitatively bad images are shown with the portions of the images that are specifically bad highlighted. In these images, variations in the muscle quality and landmarks for different individuals can be observed.When attempting this protocol, take time to understand the validity of the algorithm used by the ultrasound system. Review the anatomy of the muscle of interest and obtain sufficient practice imaging. This protocol is intended to provide pointers and describe the necessary standards.So extended field of view ultrasound may be applied to muscles beyond the two examples we provide. We expect that this method will be applied to steady muscle in both healthy and impaired populations to better understand healthy muscle function and muscle adaptation following injury or disease.

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