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  • Primer
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In vivo NMR spectroscopy

Nature Reviews Methods Primers volume 3, Article number: 91 (2023) Cite this article

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Abstract

Understanding biological processes at the molecular level is a pillar of modern science, and unique insight can be gained by studying living organisms in real time. As a non-destructive and non-targeted technique, NMR spectroscopy is uniquely capable of characterizing the chemical profile of living organisms during biochemical processes or in response to an applied stressor. In vivo NMR spectroscopy — the study of living organisms by NMR — is discussed here, including the most common and state-of-the-art experimental approaches spanning both solution-state and magic-angle spinning NMR. Key information that can be obtained and important applications — primarily monitoring biochemical processes such as growth and stress responses — are also examined. To date, in vivo NMR has been used in metabolomics studies of microorganisms, plants and invertebrates but it also has potential for medical and pharmaceutical research. Current limitations, best practices for reproducibility and optimizations are also described, including experiments and technologies capable of improving in vivo analysis. This Primer is designed to form a solid foundation for those looking to better understand or incorporate in vivo NMR studies into their own research as well as to shed light on the future of in vivo NMR.

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Fig. 1: The basis of in vivo NMR spectroscopy.
Fig. 2: Overview of the different experimental equipment and approaches used for in vivo NMR.
Fig. 3: Demonstration of the improved spectral dispersion offered by multidimensional NMR.
Fig. 4: Overview of the chemical information that can be obtained using spectral editing in CMP NMR.
Fig. 5: Advanced NMR experiments used to target specific bonds in vivo.
Fig. 6: Example uses of in vivo NMR for the study of toxicity processes.
Fig. 7: Example of in vivo NMR used to study biochemical processes.
Fig. 8: Techniques paving the future of in vivo NMR.

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Acknowledgements

The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) (Alliance (ALLRP 549399), Alliance (ALLRP 555452) and Discovery Programs (RGPIN-2019-04165)), the Canada Foundation for Innovation (CFI), the Ontario Ministry of Research and Innovation (MRI), and the Krembil Foundation for providing funding and the Government of Ontario for an Early Researcher Award.

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  1. These authors contributed equally: Daniel H. Lysak, Katelyn Downey.

Authors and Affiliations

  1. Environmental NMR Centre, Department of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada

    Daniel H. Lysak, Katelyn Downey & Andre J. Simpson

  2. Department of Chemistry, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada

    Lindsay S. Cahill

  3. Bruker BioSpin GmbH, NMR Applications, Ettlingen, Germany

    Wolfgang Bermel

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Contributions

Introduction (K.D., D.H.L., A.J.S., L.S.C. and W.B.); Experimentation (D.H.L., K.D., A.J.S., L.S.C. and W.B.); Results (D.H.L., K.D., A.J.S., L.S.C. and W.B.); Applications (D.H.L., K.D., A.J.S., L.S.C. and W.B.); Reproducibility and data deposition (K.D., D.H.L., A.J.S., L.S.C. and W.B.); Limitations and optimizations (K.D., D.H.L., A.J.S., L.S.C. and W.B.); Outlook (K.D., D.H.L., A.J.S., L.S.C. and W.B.); Overview of the Primer (K.D., D.H.L., A.J.S., L.S.C. and W.B.).

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Glossary

Chemical shift

A normalized unit that describes the position of an NMR peak on the x axis, derived from the frequency at which the nucleus resonates in an NMR spectrometer.

Coil region

The region of the radiofrequency coil where sample nuclei can be excited and detected. The NMR probe houses the detection coil. 

Cross-polarization

A phenomenon wherein polarization is transferred between different nuclei through space.

Dipolar couplings

Interactions occurring between spin-active nuclei through space.

Ex vivo

In this context, ex vivo refers to lyophilized, whole organisms that are reswollen in solution.

Peak splitting

NMR peaks are said to be ’split’ into multiplets depending on the number of other nuclei they scalar couple, resulting in characteristic peak patterns.

Planar microcoil

A microcoil where the sample is placed on a flat coil.

Presaturation

A widely used water suppression technique based on low-power irradiation of the water signal at the start of an NMR experiment.

Pro-drug

A medication that becomes active after being metabolized.

Receiver gain

An acquisition parameter that should be optimized to maximize the incoming signal without exceeding the dynamic range of the spectrometer digitizer.

Shimming

A process wherein shim coils in the spectrometer are adjusted to optimize the lineshape of a sample by compensating for inhomogeneities in the magnetic field.

Spin-active nuclei

Nuclei possessing a non-zero spin, allowing them to interact with magnetic fields and thus be detectable by NMR.

Spinning sidebands

Anisotropic peaks in an NMR spectrum that occur due to imperfect averaging of interactions due to slow spinning and appear symmetrically on either side of isotropic signals in a spectrum at a distance equal to the spin rate.

T2 filter

A filter used to remove signals from components with short T2 relaxation times such as macromolecules.

Tuning

A process in which the probe circuitry is set to the frequency at which the sample nuclei resonate.

Volume microcoil

A microcoil where the coil surrounds the sample.

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Lysak, D.H., Downey, K., Cahill, L.S. et al. In vivo NMR spectroscopy. Nat Rev Methods Primers 3, 91 (2023). https://doi.org/10.1038/s43586-023-00274-3

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