Benchtop NMR Spectroscopy of In Vivo Multicellular Organisms

Abstract

NMR spectroscopy is a critical tool for environmental and biological research, but the physical and financial barriers of standard "high-field" NMR spectrometers can limit applications, especially in the environmental sciences. Low-field benchtop NMR (¹H resonance frequencies generally ≤100 MHz) is more accessible, but its lower sensitivity and increased spectral overlap have limited the study of complex samples. Living organisms are among the most heterogeneous samples, and it is unclear if useful information can be extracted in vivo using benchtop NMR. Here, the potential of low-field (80 MHz) in vivo NMR is first assessed by analyzing ¹³C-labeling of unicellular green algae and then by monitoring a process within a multicellular organism (T. californicus). This is followed by studying live brine shrimp (A. franciscana) at ¹³C natural abundance. Adults are compared to brine shrimp cysts, with a number of spectral assignments possible and differences between the life stages clearly evident. High-field NMR is used to confirm peak assignments and provide a more comprehensive characterization of biomolecules present, ultimately making the low-field NMR data more useful. Standard experiments such as 1D ¹H, 1D ¹³C and 2D HSQC are conducted, as well as more advanced experiments such as ¹³C-SSFP, which greatly enhances ¹³C sensitivity, and reverse HSQC, which decreases spectral overlap. Ultimately, this work demonstrates that low-field NMR can effectively analyze live organisms with or without isotopic enrichment and that it holds great potential for future work, such as in vivo analysis of organisms directly in the field if/when portable NMR spectrometers become available.