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The value of preclinical diffusion MRI (dMRI) is substantial. While dMRIenables in vivo non-invasive characterization of tissue, ex vivo dMRI isincreasingly being used to probe tissue microstructure and brain connectivity.Ex vivo dMRI has several experimental advantages including highersignal-to-noise ratio and spatial resolution compared to in vivo studies, andmore advanced diffusion contrasts for improved microstructure and connectivitycharacterization. Another major advantage is direct comparison withhistological data as a crucial methodological validation. However, there are anumber of considerations that must be made when performing ex vivo experiments.The steps from tissue preparation, image acquisition and processing, andinterpretation of results are complex, with many decisions that not only differdramatically from in vivo imaging, but ultimately affect what questions can beanswered using the data. This work represents 'Part 2' of a series ofrecommendations and considerations for preclinical dMRI, where we focus on bestpractices for dMRI of ex vivo tissue. We first describe the value that ex vivoimaging adds to the field of dMRI, followed by general considerations andfoundational knowledge that must be considered when designing experiments. Wethen give guidelines for ex vivo protocols, including tissue preparation,imaging sequences and data processing including pre-processing, model-fitting,and tractography. Finally, we provide an online resource which lists publiclyavailable ex vivo dMRI datasets and dedicated software packages. In eachsection, we attempt to provide guidelines and recommendations, but alsohighlight areas for which no guidelines exist, and where future work shouldlie. An overarching goal herein is to enhance the rigor and reproducibility ofex vivo dMRI acquisitions and analyses, and thereby advance biomedicalknowledge.

Recommendations and guidelines from the ISMRM Diffusion Study Group for preclinical diffusion MRI: Part 2 -- Ex vivo imaging