Diffusion tensor imaging is a non-invasive magnetic resonance imaging technique that allows one to infer information about the directionality of brain microstructure. In this thesis an overview on diffusion imaging is presented, as well as novel algorithms and applications, with the aim of better investigating and understanding microstructural brain charachteristics and organization in vivo. In particular, a novel tractography algorithm for the evaluation of diffusion and microstructure characteristics in white matter multiple sclerosis lesions is presented, as well as a novel technique for the study of altered thalamo-cortical connectivity in traumatic brain injury patients.