This method is applied as a wrapper to existing probabilistic tractography methods. Moreover, in order to cope with the well‐known path length dependence of probabilistic tractography scores, we used a novel method called iterative confidence enhancement for tractography (ICE‐T). Therefore, we applied a representative selection of tractography techniques to ex vivo diffusion data in pig brains, involving both probabilistic and deterministic tracking algorithms, as well as local models (fitted to the data in a voxel‐wise way) based on single tensors, multiple compartments, and diffusion fiber orientation density functions (ODF). Hence, it is important to assess, using the same validation method, to what extent and under which circumstances these different methods succeed in doing so. Over time, a whole range of tractography methods have emerged that primarily aim to ameliorate these problems. However, they also demonstrated that well‐known sources of error, such as regions with crossing fibers, impact upon the performance of the tractography. Dyrby and colleagues used this technique to assess the performance of a multitensor probabilistic tractography method and showed by direct comparison (the same brain that received a tracer was also imaged) a generally high agreement between tracer and tractography results, which was reproducible across brains. However, because it is inevitable that the substance is not completely taken up by the cells and then transported down the axons, but also diffuses in intra‐axonal space, some extra calibration is needed, which is provided by a histochemical tracer. Therefore, manganese is an excellent means of validating diffusion tractography. On the other hand, in vivo manganese tracers can be visualized by magnetic resonance imaging (MRI) and thereby used to characterize the connectivity of an injection site with the entire brain, which is very comparable to the connectivity profile of a seed region obtained by tractography. For example, in a recent study Jbabdi and colleagues successfully used tracers for long‐range connections of the ventral prefrontal cortex in macaque monkeys and compared this to ex vivo tractography. Although classical in vivo tracing techniques in animals yield very accurate quantitative estimates of the connectivity, they can typically only be applied to just one injection site per animal, and require painstaking procedures. However, such a ground truth regarding the fiber connectivity within the entire brain is very difficult to obtain. In order to assess the confidence we may place in results from different tractography methods and to gain deeper insight into the nature of the various problems, validation against a “ground truth” would be invaluable. While the resulting fiber pathways certainly contain valuable information about the underlying architecture, they are subject to a number of serious pitfalls and limitations. Tractography is a class of techniques that aim to extract information about white matter fiber systems from diffusion‐weighted magnetic resonance imaging (dwMRI) data. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc. In particular, the increase of spatial resolution, under preservation of the signal‐to‐noise‐ratio, is key. Instead, the crucial challenge in making tractography a truly useful and reliable tool in brain research and neurology lies in the acquisition of better data. Closer inspection of the results led to the conclusion that these problems mainly originate from regions with complex fiber arrangements or high curvature and are not easily resolved by sophisticated local models alone. However, false positive connections were very common and, in particular, we discovered that it is not possible to achieve high sensitivity (i.e., few false negatives) and high specificity (i.e., few false positives) at the same time. While the voxel‐wise agreement was very limited, qualitative assessment revealed that tractography is capable of finding the major fiber tracts, although there were some differences between the methods. A representative selection of tractography methods were compared to manganese tracing on a voxel‐wise basis, and a more qualitative assessment examined whether, and to what extent, certain fiber tracts and gray matter targets were reached. In this study, we used invasive tracing to evaluate white matter tractography methods based on ex vivo diffusion‐weighted magnetic resonance imaging (dwMRI) data.
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