Considerations and recommendations from the ISMRM Diffusion Study Group for preclinical diffusion MRI: Part 3-Ex vivo imaging: Data processing, comparisons with microscopy, and tractography.

Preclinical diffusion MRI (dMRI) has proven value in methods development and validation, characterizing the biological basis of diffusion phenomena, and comparative anatomy. While dMRI enables in vivo non-invasive characterization of tissue, ex vivo dMRI is increasingly being used to probe tissue microstructure and brain connectivity. Ex vivo dMRI has several experimental advantages that facilitate high spatial resolution and high SNR images, cutting-edge diffusion contrasts, and direct comparison with histological data as a methodological validation. However, there are a number of considerations that must be made when performing ex vivo experiments. The steps from tissue preparation, image acquisition and processing, and interpretation of results are complex, with many decisions that not only differ dramatically from in vivo imaging of small animals, but ultimately affect what questions can be answered using the data. This work concludes a three-part series of recommendations and considerations for preclinical dMRI. Herein, we describe best practices for dMRI of ex vivo tissue, with a focus on image pre-processing, data processing, and comparisons with microscopy. In each section, we attempt to provide guidelines and recommendations but also highlight areas for which no guidelines exist (and why), and where future work should lie. We end by providing guidelines on code sharing and data sharing and point toward open-source software and databases specific to small animal and ex vivo imaging.