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Figure 1.10 Microstructural reorganization quantified by diffusion-weighted imaging (DWI) during epileptogenesis predicts disease progression. (A1–6) Representative coronal sections from diffusion-weighted tractography at different time points during epileptogenesis (before injection = pre; 1 day, 4 days, 7 days, 14 days, and 31 days following SE). (B1–6) Enlarged images. (C–D) Representative tractography image and a Nissl-stained section of corresponding brain regions for anatomical comparison. Computed fibers relate to major axonal pathways and brain regions exhibiting highly oriented dendrites (cc, corpus callosum). (E, G) Enlarged tractography images demonstrating the distinct orientation of streamlines in different hippocampal layers. (F, H) Corresponding 4′,6-diamidino-2-phenylindole (DAPI)-stained sections. Scale bars in A, 2 mm; B–D, 500 mm; H (left), 100 mm (I, K, M, O). Quantitative analysis of DWI metrics in the dentate gyrus (DG), plotted for individual mice (left panel; controls, black, n = 5; kainate-injected animals color-coded) and for groups during epileptogenesis. [42] Philipp Janz et al. (2017), figure 05[p.10]/eLife Sciences Publications Ltd./CC BY 4.0.

      1.4 Augmented MR Microscopy

In situations in which microscopy is desired of objects that themselves have small dimensions, then there is an MR sensitivity advantage when using micro detectors. The challenge of sample handling at these dimensions, often achieved by microfluidic systems, can be turned into a feature when one considers the library of lab-on-a-chip (LOC) technologies already available, offering additional degrees of freedom in terms of sample management and interaction. Integration of NMR and microfluidics has continued to advance, starting from the earliest microcoil reports [43,44] to recent efforts taking advantage of LOC principles to enable microfluidic-based perfusion [35,45–49], electrochemistry [50], and hyperpolarization [51–53]. As discussed extensively in this chapter, micro-NMR coupled with MR microscopy is established and continues to evolve, with one branch of this evolution now extending toward fully exploring the augmented micro-MR system to further enhance the applicability of MR microscopy.

      1.4.1 Perfusion

      Perfusion can be considered a subclass of flow-based methods. In the context of this discussion, the definition of perfusion is relaxed slightly to include the passage of a fluid through microfluidic systems for the purpose of transporting nutrients and waste. Therefore, such systems can be used to maintain biological samples under conditions conducive to normal behavior, enabling long-term measurements of the system under normal and stimulated situations. Systems may include cell populations/layers/clusters and may increase in complexity up to tissue slices, organ-on-a-chip, and small organisms. Long-term measurement of such systems while using MR-compatible technical systems enables spatially resolved, longitudinal monitoring of morphology as a function of interesting stresses.

Starting from spectroscopy, perfusion-enabled microfluidic devices for long-term monitoring of biological systems have been used to monitor metabolic flux. Kalfe et al. [49] monitored a single tumor spheroid with diameter 500 µm over 24 h to characterize the transition from oxidative to glycolytic metabolism. Given the small volumes, it is important to ensure that the metabolic activity is reflective of the actual biological system and not perturbed, for example, by a lack of oxygen in the culture medium. This issue has been directly addressed by Yilmaz and Utz [47], who used a gas permeable membrane in combination with a micro-stripline [46] to ensure adequate oxygen supply for in situ cell culture NMR spectroscopy. Using 3D printing to produce MR-compatible microfluidic systems Montinaro et al. [45], demonstrated spectroscopy of small organisms, achieving detection volumes of 100 pl and therefore substructure spectroscopy of Caenorhabditis elegans in their work.

Incubator systems have been implemented for improved tissue slice MR microscopy. To enable long-term microscopy of a biologically viable tissue, incubator systems must not only manage perfusion but also gas concentration and temperature control. Flint et al. [48]. developed such an incubator system compatible with a 600-MHz NMR spectrometer. They demonstrated diffusion-weighted imaging of rat cortical slices with 31.25 µm isotropic resolution (1.5 h measurement time) over 21 h. The challenge for soft tissue incubation in vertical bore NMR systems is preventing tissue deformation caused by gravity. This can be addressed, for example, by physically clamping the tissue taking care not to unnecessarily perturb the tissue function. This challenge can be circumvented by ensuring gravity is perpendicular to the tissue surface, easily achieved in horizontal bore systems. Kamberger et al. [35] implemented an incubator for mouse brain slice imaging under this condition, with the added feature of a LL for magnetic field-focusing and improved SNR [33]. Using a 9.4-T MRI system, T1-weighted images could be obtained in 8 min with 0.5 mm slice thickness and in-plane resolution of 0.1 × 0.1 mm, importantly, with a factor of 10 improved SNR yielded by the LL (Figure 1.7). In a clever use of capillary forces, tissue–air interfaces perpendicular to B₀ were avoided by allowing the perfusion medium to slightly overfill the tissue chamber thereby eliminating magnetic susceptibility-induced imaging artifacts.

      1.4.2 Electrochemistry

The microfluidic LOC and micro total analysis system (µTAS) communities have recognized the added value of implementing electro-manipulation capabilities. From the perspective of biological samples, the electrical degree of freedom enables new methods for sample manipulation and sample analysis [54]. From a chemical perspective, electric fields can be used for selective analyte transport or to drive electrochemical reactions. Simultaneous integration with microscopy has the potential to spatially localize the electro-response of the system, noninvasively and label-free in the case of MR microscopy.

MR-coupled electrochemical methods are often used to study electrophoretic behavior of electrolytes [55–57]. In these experiments, the electrodes used to generate the electric field are located outside of the NMR-sensitive volume. In the case of monitoring electrochemical reactions, it would be advantageous to localize the reaction within or in the immediate vicinity of the NMR detection volume. This has been demonstrated outside of the microfluidic domain, with recent efforts aiming to retain high-resolution NMR spectra at high magnetic fields using standard sample tubes [58–61]. NMR-compatible microfluidic systems with integrated electrodes are rare; in one example, a digital microfluidic approach has been demonstrated as a means to manipulate droplets and deliver sample to the NMR detection volume [62,63]. An in situ electrochemical system has recently been reported, including an analysis of the appropriate electrode properties that maintain acceptable NMR spectral resolution [50] (Figure 1.11). In this report, MR microscopy is additionally demonstrated, although in this case for the purpose of B₀ and B₁ field characterization.