Aiming to improve noninvasive assessment of tissue microstructure, a physics-based simulation of diffusion tensor imaging (DTI) was used to compare helix angles between DTI and structure tensor (ST) analyses of a confocal myocardial image stack. Rodent myocardium was imaged using extended volume confocal microscopy producing a high-resolution image volume, which was segmented into intracellular and extracellular compartments. The image volume was divided into voxel-blocks, which served as the DTI simulation voxels. The DTI acquisition showed good agreement with both the ST and manually measured helix angles. This proof-of-concept work demonstrates the feasibility of direct comparison of confocal images with DTI.