Sub-Microliter ¹H Magnetic Resonance Spectroscopy for In Vivo High-Spatial Resolution Metabolite Quantification in the Mouse Brain

Proton magnetic resonance spectroscopy (MRS) offers a non-invasive, repeatable, and reproducible method for in vivo metabolite profiling of the brain and other tissues. However, metabolite fingerprinting by MRS requires high signal-to-noise ratios for accurate metabolite quantification, which has traditionally been limited to large volumes of interest, compromising spatial fidelity. In this study, we introduce a new optimized pipeline that combines LASER MRS acquisition at 11.7 T with a cryogenic coil and advanced offline pre- and post-processing. This approach achieves a signal-to-noise ratio sufficient to reliably quantify 19 distinct metabolites in a volume as small as 0.7 μL within the mouse brain. The resulting high spatial resolution and spectral quality enable the identification of distinct metabolite fingerprints in small, specific regions, as demonstrated by characteristic differences in N-acetylaspartate, glutamate, taurine, and myo-inositol between the motor and somatosensory cortices. We demonstrated a decline in taurine and glutamate in the primary motor cortex between 5 and 11 months of age, against the stability of other metabolites. Further exploitation to cortical layer-specific metabolite fingerprinting of layer I–III to layer VI–V in the primary motor cortex, with the latter showing reduced taurine and phosphoethanolamine levels, demonstrates the potential of this pipeline for detailed in vivo metabolite fingerprinting of cortical areas and subareas. (Figure presented.)