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Cardiac disease, such as hypertrophic cardiomyopathy, is often accompanied by myocardial fiber disarray. Echocardiography has been of significant use for the clinical diagnosis of the cardiac function but not for the measurement of myocardial fiber structure yet. In this study, we thus proposed a novel ultrasound-based shear wave imaging (SWI) method to noninvasively assess myocardial fiber orientation, in comparison with histology and diffusion tensor imaging (DTI).Fresh porcine myocardial samples (n=6; 20x20x30 mm3) were studied in vitro. Known in physics, shear wave propagates faster along than across the fibers. SWI is such a technique that utilizes focused ultrasound beams to generate shear waves propagating at velocities between 1 and 10 m/s in different directions with respect to each fiber layer throughout the entire myocardial thickness (10-25 mm). A prototype ultrasound scanner equipped with an 8 MHz linear array probe was employed. The fiber angle at each myocardial depth was determined by the shear wave propagation direction where the maximum shear wave speed occurred. Fiber angles were defined between -90° and 90° with 0° denoting fibers aligned with the local circumferential axis of the heart. Both the myocardial sample and the ultrasound probe were secured in a customized device compatible with the magnetic resonance imaging scanner (7T, Bruker) to assure imaging co-registration. In DTI, diffusion was encoded in six directions. A total of 270 diffusion-weighted images (b = 1000 s2/mm, FOV=30 mm, Matrix size= 64x60, TR=9 s, TE=19 ms, 24 averages) and 45 b0 images were acquired in 13 h and were further analyzed using a freeware, MedINRIA. Histology with H&E staining was performed and analyzed using optical microscopy to measure the transmural fiber orientation.The transmural fibers in the in vitro porcine mid-anterior myocardial region assessed by SWI gradually oriented from +80° (upper-right to bottom-left) at the endocardium to -50° (upper-left to bottom-right) at the epicardium. This result was not only in good agreement with the literature but also well correlated with histology (r2=0.91±0.02, p<0.001). Moreover, good correlation between SWI and DTI fiber angle estimates was found (r2=0.83±0.01, p<0.0001).We have demonstrated that ultrasound-based SWI was capable of mapping transmural fiber orientation and provided measurements comparable to histology and DTI. In vivo feasibility of SWI in mapping the fiber orientation is being examined. SWI may serve as a new diagnostic tool for the evaluation of myocardial structure and its associated cardiac pathologies.