Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging

Nat Protoc. 2021 Jul;16(7):3298-3321. doi: 10.1038/s41596-021-00538-w. Epub 2021 Jun 2.


Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks. Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution. This is currently not possible with any other imaging technique. The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1-2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation. The specificity of the chemical reaction can also help users identify unknown chemical identities. Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / diagnostic imaging*
  • Brain / metabolism*
  • Limit of Detection
  • Male
  • Neurotransmitter Agents / metabolism*
  • Optical Imaging*
  • Rats
  • Rats, Sprague-Dawley
  • Reference Standards
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Neurotransmitter Agents