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 EFOV-US also exist18,20,30. A linear array transducer 14L5 (frequency bandwidth 5-14 MHz) was utilized. The muscles imaged in this protocol are just a small subset of muscles for which US images have been captured and fascicle lengths measured (e.g., triceps25, extensor carpi ulnaris23, medial gastrocnemius10, vastus lateralis24, biceps femoris8,31). This protocol is intended to provide pointers and describe the necessary standards so that that it may be applied to muscles beyond the two examples we provide.
1. Collecting EFOV-US images of muscles
Preparation
<ol>
	<li>Sonographer Preparation
	<ol>
		<li>Prior to operating the ultrasound system, read through the system&#8217;s manual to become familiar with system safety, care for maintaining the system, system setup and controls, etc. In addition, review the system&#8217;s instructions for obtaining EFOV-US images and be familiar with the method implemented to obtain the EFOV-US images. 
		NOTE: Different ultrasound systems name the EFOV-US mode using different terminology. For example, in the system used here, the EFOV mode is referred to as &#8220;Panoramic Imaging&#8221;. While the technical details of the algorithm implemented in various commercial systems are usually intellectual property and therefore not freely available, from a cursory review, many commercial systems with panoramic ultrasound capabilities describe an approach similar to the one described by Weng et al.22. Evaluating the general validity of measurements acquired from any system, either by obtaining more detailed information directly from the company who manufactures the system, by using an imaging phantom26,32, or by other means (e.g., comparison to animal dissection24) is recommended as an important step before initiating research involving human participants.</li>
		<li>Take time to become familiar with the anatomy of the muscle(s) of interest as well as the surrounding anatomy. It is suggested that the sonographer use an anatomy textbook or preferably an interactive online 3D anatomy model to become familiar with the anatomy of interest.</li>
	</ol>
	</li>
	<li>Participant Preparation
	<ol>
		<li>Explain the protocol of the study to the participant and acquire IRB approved consent prior to beginning the imaging protocol.</li>
		<li>Ask the participant to wear appropriate clothing to enable access to the muscle of interest. For example, if the sonographer plans to image a forearm muscle, the participant should be asked to wear a short-sleeved shirt.</li>
		<li>Seat the participant in an adjustable chair that can be locked in place. Take time to adjust the chair to make the participant as comfortable as possible while still providing access to the muscle of interest. 
		NOTE: If an adjustable chair which can lay completely flat is not available, some study designs may require the use of a table to access the muscle of interest (i.e., hamstrings).</li>
		<li>Place the joint(s) that the muscle of interest spans in a posture that can be controlled and repeated. Use clinical guidance33 for locating anatomical landmarks and implementing goniometry; use ISB standards for defining the joint coordinate system34,35. In general, to measure joint angle, mark anatomical landmarks with skin safe marker (Table of Materials) and then align the center of a handheld goniometer up with the axis of rotation of the joint and the arms of the goniometer up with the joint segments. 
		NOTE: If imaging passive muscle, placing the muscle of interest in a relatively lengthened position is recommended to avoid imaging slack muscle.
		<ol>
			<li>To replicate&#160;the biceps brachii as imaged in this study, seat participants with feet supported, back straight, shoulder at 85&#176; of abduction and 10&#176; of horizontal flexion, elbow at 25&#176; flexion, and forearm, wrist, and fingers at neutral.</li>
			<li>To replicate the tibialis anterior as imaged in this study, seat participants with knee at 60&#176; of flexion and the ankle at 15&#176; of plantar flexion.</li>
		</ol>
		</li>
		<li>Secure the participants limb using cloth straps to minimize movement during the imaging protocol.</li>
	</ol>
	</li>
</ol>
Image Acquisition
<ol start="3">
	<li>Plug in and turn on the ultrasound system. Ensure that the exam is set to Musculoskeletal, the transducer in&#160;use is selected (here we used 14L5), and the transmit frequency is set between 5-17 MHz (here 11MHz was used), a typical frequency range for musculoskeletal imaging. Higher frequencies are generally used for more superficial imaging as they improve resolution but decrease wave penetration.</li>
	<li>Go into the system settings to adjust the footswitch settings. For the purposes of this protocol, we recommend setting the footswitch to start/stop the imaging. If the footswitch in use has multiple pedals, set additional pedals to &#8220;Freeze&#8221; or &#8220;Pause&#8221;, and &#8220;Print&#8221; or &#8220;Store&#8221; the image.</li>
	<li>Apply a generous amount of ultrasound gel to the head of the transducer.</li>
	<li>Place the transducer on the participant&#8217;s skin on the approximate region of interest.</li>
	<li>Move the transducer in the short axis plane of the muscle. Note that the transducer has a small protuberance on one side, called an indicator. The side of the transducer that has the indicator corresponds with the left side of the ultrasound image. When imaging in the short axis, have the sonographer keep the indicator pointed laterally and when the sonographer is in long axis, point the indicator distally.</li>
	<li>Identify the muscle of interest in the short axis plane (perpendicular to muscle fiber direction) and move the transducer distal and proximal to get a full visualization of the muscle path.
	<ol>
		<li>Mark important anatomical landmarks (i.e., the lateral and medial edges of the muscle, the muscle tendon junction, and muscle insertion) using skin safe ink markers (Table of Materials).</li>
	</ol>
	</li>
	<li>Once the location of the muscle has been identified and properly marked, have the sonographer move the ultrasound transducer in the long axis plane (parallel to the muscle fiber direction).</li>
	<li>Beginning at either the distal or proximal end of the muscle, rotate and tilt the transducer to identify the fascicle plane at that point. Make a mark on the skin when the correct transducer position has been established.</li>
	<li>Once the approximate fascicle plane has been established along the entire desired length to be scanned, have the sonographer practice following this path.</li>
	<li>To begin collecting images, put the ultrasound system in EFOV-US mode.</li>
	<li>Starting at one end of the muscle, click the footswitch to start image acquisition and slowly and continuously move the ultrasound transducer in the long axis. Once the end of the muscle has been reached, click the footswitch to end image acquisition.</li>
	<li>Practice and ensure the correct transducer path. This may take several practice images before consistently obtaining &#8220;quality&#8221; EFOV-US images (See section 2 for explanation of quality images).</li>
	<li>To optimize image visibility and clarity, consider adjustments to the following parameters.
	<ol>
		<li>Depth: If image acquisition ends before the desired length of the muscle can be captured, increase the depth of the image (in the system used here, increasing image depth increases the absolute length the scan can be).</li>
		<li>Focus: Place the focus arrow in the lower half of the Image just below the muscle of interest.</li>
		<li>Gain: Ensure the gain is balanced through the depth of the image.</li>
		<li>Speed: Image at the optimum speed as guided by the indicator (in most systems a speed indicator displays on the monitor during panoramic imaging).</li>
	</ol>
	</li>
	<li>Once qualitatively good images have been collected (step 2.1), hit the Print/Store footswitch pedal or a synonymous button on the control panel to save the image.</li>
	<li>Repeat steps 1.13-1.16 until 3 quality EFOV-US images of the muscle are obtained.</li>
	<li>Repeat steps 1.6-1.17 until all muscles of interest are obtained.</li>
	<li>Use a towel to gently wipe the gel from the participant&#8217;s skin. Then have the participant rinse off the area of the skin or use a damp towel to wipe the skin that was exposed to the gel. Dry.</li>
	<li>Wipe gel from the head of the transducer and disinfect.</li>
	<li>Export images as uncompressed DICOM images onto a CD-DVD, flash drive, or through the local network onto a computer.</li>
</ol>
2. Determining &#8220;quality&#8221; of the EFOV-US image
<ol>
	<li>Following step 1.13, have the sonographer identify and evaluate the quality of key anatomical features of the muscle of interest and its surrounding anatomy. This is a qualitative assessment based on the sonographer&#8217;s knowledge of anatomy and musculoskeletal tissue echogenicity (ability of a tissue to reflect ultrasonic waves). For an EFOV-US images to be considered qualitatively &#8220;good&#8221; the following should be met:
	<ol>
		<li>In any long-axis image of a muscle, check that the sonographer can clearly identify the muscle as a hypoechoic (dark) shape with hyperechoic (bright) boundaries which represent the deep and superficial muscle fascia.</li>
		<li>Between the muscle boundaries, check that the sonographer can identify the connective tissue surrounding a muscles fascicle as hyperechoic (bright) lines. 
		NOTE: When imaging multi-pennated muscles, the image should also contain central tendon(s) that show up in the muscle belly, between the deep and superficial muscle fascia, as a hyperechoic (bright) structure.</li>
		<li>Check that the image does not have excessive bending. This is usually indicated by shadows or gaps in the image or a jagged flexible ruler line over the image.</li>
	</ol>
	</li>
	<li>If the image is missing one or more of the tissue structures described in 2.1, deem the image &#8220;qualitatively poor&#8221; and return to live 2D-mode.</li>
</ol>
3. Quanitfying Muscle Fascicle Length
<ol>
	<li>To quantify muscle fascicle length, use ImageJ, an open source image processing platform. ImageJ can be downloaded at https://imagej.net/Downloads. 
	NOTE: Though ImageJ is frequently implemented24,25,31,36,37,38, quantification of muscle fascicle length may be measured using other image processing software8,39 or custom codes40,41.</li>
	<li>Once downloaded, open the ultrasound images as DICOM images in ImageJ by clicking File | Open and selecting the image to analyze.</li>
	<li>To ensure that the DICOM image properties have been preserved, click on the Straight Line tool in the Tools menu and draw a straight line from 0 to 1 cm on the ruler on the side of the ultrasound image. Then go to Analyze | Measure to measure the line made. If the image properties have been preserved, the length of the straight line should be 1 cm.</li>
	<li>To measure fascicle lengths in the image, complete the following.
	<ol>
		<li>Right click on the Straight Line tool.</li>
		<li>Select Segmented Line.</li>
		<li>Move the cursor onto the image and click at one end of the fascicle that has been chosen to be measured. 
		NOTE: Only make measurements on fascicles that the entire fascicle path (i.e., from one aponeurosis to the next aponeurosis or aponeurosis to central tendon) can convincingly be seen.</li>
		<li>Click along the path to ensure curvature in the fascicle path is captured.</li>
		<li>Once the end of the fascicle path is reached, double click to end the line and go to Analyze | Measure to measure the length of the line. 
		NOTE: A new window, &#8220;Results&#8221;, will pop up the first time a measurement is made. What values are displayed can be managed in the Results window by going to Results | Set Measurements.</li>
	</ol>
	</li>
	<li>Repeat steps 3.4.3-3.4.5 until multiple fascicle measures are made in a single image.</li>
	<li>Save fascicle measurements by clicking File | Save on the results tab or the values can be copy and pasted into another document/spreadsheet.</li>
</ol>

The authors have nothing to disclose.

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 &amp;amp;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 ...

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3.6K Views.  Northwestern University. 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.

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Developmental Biology

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Bioengineering

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

Ultrasound Evaluation of Achilles Tendon Morphology

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.

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.

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

Ultrasound Protocol for Perioperative Frailty Assessment

Identifying Frailty Using Point-of-Care Ultrasonography: Image Acquisition and Assessment

Frailty is a significant predictor of a range of adverse outcomes in surgical patients, including increased mechanical ventilation time, longer hospital stays, unplanned readmissions, stroke, delirium, and death. However, accessible tools for screening in clinical settings are limited. Computed tomography of the psoas muscle is the current standard imaging device for measuring frailty, but it is expensive, time-consuming, and exposes the patient to ionizing radiation. Recently, the use of point-of-care ultrasound (POCUS) has emerged as a potential tool to determine the presence of frailty and has been shown to accurately predict frailty and postoperative outcomes. In this article, we will describe the image acquisition of the quadriceps muscles and explain how they can be used to determine frailty and predict postoperative adverse events. We will present information on probe selection, patient positioning, and troubleshooting. Images from a demonstration will be used to present the POCUS technique and example results. The article will culminate in a discussion of the use of these images in medical decision-making and potential limitations.

Author Spotlight: Assessing Surgical Frailty with Point-of-Care Ultrasound of Quadriceps Muscles

Here, we present a protocol to assess frailty in the perioperative setting using point-of-care ultrasound to measure quadriceps thickness. This method offers a practical, non-invasive alternative to traditional assessment methods, potentially enhancing perioperative care by quickly identifying frail patients.

Frailty is a significant predictor of a range of adverse outcomes in surgical patients, including increased mechanical ventilation time, longer hospital ...

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

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

Summary

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A Novel Application of Musculoskeletal Ultrasound Imaging

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Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

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Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Measurement of Healthy and Injured Triceps Surae Morphology

Identifying Frailty Using Point-of-Care Ultrasonography: Image Acquisition and Assessment

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography