Disentangling the effects of restriction and exchange with diffusion exchange spectroscopy

Diffusion exchange spectroscopy (DEXSY) is a multidimensional NMR technique that can reveal how water molecules exchange between compartments within heterogeneous media, such as biological tissue. Data from DEXSY experiments is typically processed using numerical inverse Laplace transforms (ILTs) to produce a diffusion-diffusion spectrum. A tacit assumption of this ILT approach is that the signal behavior is Gaussian - i.e., the spin echo intensity decays exponentially with the degree of diffusion weighting. The assumptions that underlie Gaussian signal behavior may be violated, however, depending on the gradient strength applied and the sample under study. We argue that non-Gaussian signal behavior due to restrictions is to be expected in the study of biological tissue using diffusion NMR. Further, we argue that this signal behavior can produce confounding features in the diffusion-diffusion spectra obtained from numerical ILTs of DEXSY data - entangling the effects of restriction and exchange. Specifically, restricted signal behavior can result in broadening of peaks and in the appearance of illusory exchanging compartments with distributed diffusivities, which pearl into multiple peaks if not highly regularized. We demonstrate these effects on simulated data. That said, we suggest the use of features in the signal acquisition domain that can be used to rapidly probe exchange without employing an ILT. We also propose a means to characterize the non-Gaussian signal behavior due to restrictions within a sample using DEXSY measurements with a near zero mixing time or storage interval. We propose a combined acquisition scheme to independently characterize restriction and exchange with various DEXSY measurements, which we term Restriction and Exchange from Equally-weighted Double and Single Diffusion Encodings (REEDS-DE). We test this method on ex vivo neonatal mouse spinal cord - a sample consisting primarily of gray matter - using a low-field, static gradient NMR system. In sum, we highlight critical shortcomings of prevailing DEXSY analysis methods that conflate the effects of restriction and exchange, and suggest a viable experimental approach to disentangle them.