Cardiac architecture is fundamental to cardiac function and can be assessed non-invasively with diffusion tensor imaging (DTI). Here, we aimed to overcome technical challenges in ex vivo DTI in order to extract fine anatomical details and to provide novel insights in the 3D structure of the heart. An integrated set of methods was implemented in ex vivo rat hearts, including dynamic receiver gain adjustment, gradient system scaling calibration, prospective adjustment of diffusion gradients, and interleaving of diffusion-weighted and non-diffusion-weighted scans. Together, these methods enhanced SNR and spatial resolution, minimised orientation bias in diffusion-weighting, and reduced temperature variation, enabling detection of tissue structures such as cell alignment in atria, valves and vessels at an unprecedented level of detail. Improved confidence in eigenvector reproducibility enabled tracking of myolaminar structures as a basis for segmentation of functional groups of cardiomyocytes. Ex vivo DTI facilitates acquisition of high quality structural data that complements readily available in vivo cardiac functional and anatomical MRI. The improvements presented here will facilitate next generation virtual models integrating micro-structural and electro-mechanical properties of the heart.
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