Diffusion tensor imaging (DTI) is increasingly used to investigate the 3D tissue architecture in the heart. It enables estimation of the principal orientations of cells and sheets based on the principal eigenvectors ν₁, ν₂ and ν₃. However, the process of diffusion in biological tissue is complicated by the presence of cell membranes, organelles and metabolites. Independent validation is critical to improving interpretation of DTI data, and broadening its application. Previous validation studies made use of ink prints¹ and microscopic histology.^2,3 However, these 2D approaches are prone to tissue distortion during sample preparation and are destructive. Reconstructing the data in 3D in the presence of distortions remains an unresolved challenge. Methods for extracting tissue structural information in 3D include anatomical MRI⁴ and CT with X-ray laboratory sources⁵ which are limited in resolution, scanning electron microscopy⁶ and confocal microscopy⁷ which are limited in field-of-view (FOV), and synchrotron imaging (SI) which strikes an excellent balance between resolution and coverage.^8,9 Here, we investigate for the first time, the prospect of validating DTI with structure tensor (ST) analysis of SI data.One heart was excised from a healthy female Sprague-Dawley rat, fixed in isosmotic Karnovsky’s fixative, doped with 2mM Gd and embedded in a tube of agarose gel for MRI and SI. Non-selective 3D fast spin echo DTI data were acquired on a 9.4 T preclinical MRI scanner (Agilent, CA, USA) with a transmit-receive birdcage coil (Rapid Biomedical, Rimpar, Germany) of inner diameter = 20 mm. TR / TE1 = 250 / 9.5 ms, echo spacing = 5.1 ms, echo train length = 8, FOV = 21.6 x 14.4 x 14.4 mm, resolution (isotropic) = 100 μm, number of non-DW images = 4, number of DW directions = 30, b = 1,000 s/mm². SI was subsequently performed at beamline I13-2 (imaging branch) of the Diamond Light Source (Didcot, UK) using a polychromatic beam with X-ray energies in the interval 20 - 30 keV. Data were acquired with (A) 1.25X objective, FOV_{reconstructed} = 14.0 x 14.0 x 9.6 mm, pixel size = 3.6 μm, and (B) 4X objective, FOV_{reconstructed} = 4.5 x 4.5 x 3.0 mm, pixel size = 1.1 μm. Diffusion tensors were fitted to the DTI data, and diffusion primary eigenvectors (ν_1,DT) were mapped. STs were calculated based on the signal gradient intensity in the SI absorption data, and transformed such that the primary eigenvectors (ν_1,ST) were oriented along the dominant cell long axes. Helix angles were calculated as the elevation angle of ν_1,DT and ν_1,ST perpendicular to the axial plane. All data were analysed with custom code in Matlab R2013A (Mathworks, Natick, USA).Figure 1 depicts (A) diffusion primary eigenvector (ν_1,DT) maps and (B) SI absorption data in a short-axis mid-myocardial slab. The data exhibit substantial 3D coverage, isotropic image resolution and excellent anatomical correspondence at the macroscopic scale free from distortion. Closer inspection of ν_1,DT in a 3D region-of-interest (ROI), as in Figure 2A, reveals the known transmural variation in helix angle, with the color scheme encoding for ν_1,DT orientation. Figure 2B illustrates the superior resolution of SI, and its ability to resolve cellular structures. Diffusion and structure tensors in 2D ROIs across the septal wall (Figure 3) are in good agreement, and recapitulate the transmural variation in myocyte orientation, where helix angles progress from negative to positive values from the subepicardium to the subendocardium.DTI is a valuable method that enables inference of 3D tissue structure in the whole intact organ. Previous efforts to validate DTI using other high resolution imaging methods are routinely limited by FOV or resolution, or are susceptible to tissue distortion during sample preparation. SI overcomes these constraints. In conjunction with ST analysis, it has both the resolution for assessing cellular microstructure, and the coverage for measuring macroscopic fibre and sheet architecture in the whole heart. Synchrotron radiation is known for its high brilliance, and provides orders of magnitude higher flux than X-ray laboratory sources, enhancing the achievable resolution. ST-SI can readily yield tensors at a higher resolution than DTI. This would help disentangle complex structural information that would have been averaged over the larger voxels used in DTI. ST-SI is uniquely placed to serve to validate and aid development of DTI and advanced diffusion MRI methods, and also to inform mechanical modelling of biological tissue. In further work, we will obtain SI data in additional hearts with whole organ coverage, optimize the acquisition parameters and improve tissue contrast via phase retrieval.