An ultrasound test was performed on 4 lower extremity muscles: rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG) using a LOGIQ e ultrasound-imaging device (GE Healthcare UK Ltd., Chalfont, Buckinghamshire, England). The dominant leg was tested. Participants were examined while resting supine on an examining table. Ultrasound Brightness mode (B-mode) with musculoskeletal scanning preset and a multi-frequency linear transducer (8-12 MHz) with 12.7 × 47.1mm footprint were used. The beam width of the transducer was approximately 2.0mm at its narrowest point. Gain and transducer frequency were adjusted to 58-dB and 8 MHz, respectively. Scanning depth was set to 4 cm with an apparent spatial resolution of 80 μm/pixel. The scanning depth was only increased when testing participants with greater subcutaneous fat to allow for capturing enough muscle area. Time gain compensation was adjusted to neutral position. Focus number and area were increased to maximum and kept consistent across all participants to adjust for differences in muscle size among participants. Other ultrasound settings were unchanged from the preset.
Before starting the ultrasound study, the upper and lower leg length of each participant was measured from the superior lateral aspect of the patella to the anterior superior iliac spine and from the inferior lateral aspect of the patella to the calcaneus, respectively. Marks were made on the anterior and posterior parts of the 1/3 and 1/4 of upper and lower leg length, measured from the patella. The purpose of the marks was to ensure that the scanning locations between ultrasound and MRI as well as between participants were consistent. A generous amount of ultrasound gel was applied to avoid excessive pressure on the skin. Each scan involved a 16-second ultrasound clip on 1 of the marks, and each muscle was scanned twice (both 1/3 and 1/4 marks). A total of 8 scans were obtained from each participant. Each ultrasound clip was reviewed, and 1 frame with the best focus was chosen and saved into a JPEG image for analysis. Muscle EI was determined by gray-scale analysis using ImageJ16 . A muscle of interest was circled manually while avoiding surrounding fascia and bone. The mean voxel intensity of the selected muscle region was obtained from each measurement, and an average of 3 measurements was calculated. Subcutaneous fat thickness, muscle thickness, and area of the muscle of interest were also recorded. Images were analyzed by 2 investigators to test for the inter-rater reliability.
Before starting the ultrasound study, the upper and lower leg length of each participant was measured from the superior lateral aspect of the patella to the anterior superior iliac spine and from the inferior lateral aspect of the patella to the calcaneus, respectively. Marks were made on the anterior and posterior parts of the 1/3 and 1/4 of upper and lower leg length, measured from the patella. The purpose of the marks was to ensure that the scanning locations between ultrasound and MRI as well as between participants were consistent. A generous amount of ultrasound gel was applied to avoid excessive pressure on the skin. Each scan involved a 16-second ultrasound clip on 1 of the marks, and each muscle was scanned twice (both 1/3 and 1/4 marks). A total of 8 scans were obtained from each participant. Each ultrasound clip was reviewed, and 1 frame with the best focus was chosen and saved into a JPEG image for analysis. Muscle EI was determined by gray-scale analysis using ImageJ16 . A muscle of interest was circled manually while avoiding surrounding fascia and bone. The mean voxel intensity of the selected muscle region was obtained from each measurement, and an average of 3 measurements was calculated. Subcutaneous fat thickness, muscle thickness, and area of the muscle of interest were also recorded. Images were analyzed by 2 investigators to test for the inter-rater reliability.