Label-Free Ratiometric Imaging of Serotonin in Live Cells

ACS Chem Neurosci. 2017 Nov 15;8(11):2369-2373. doi: 10.1021/acschemneuro.7b00132. Epub 2017 Aug 24.


Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations using specially designed fluorescent labels. We describe a label-free ratiometric imaging technique for direct detection of changes in intravesicular serotonin concentration in live cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using relatively benign three-photon excitation and split it into two imaging channels, whose ratio reports the concentration. The technique is sensitive at a physiologically relevant concentration range (10-150 mM serotonin). As a proof of principle, we measure the increase of intravesicular serotonin concentration with the addition of external serotonin. In general, since emission spectra of molecules are often sensitive to concentration, our method may be applicable to other natively fluorescent intracellular molecules which are present at high concentrations.

Keywords: Multiphoton microscopy; neurotransmitter imaging; quantitative imaging; serotonergic neurons.

MeSH terms

  • Animals
  • Calibration
  • Cell Line
  • Image Processing, Computer-Assisted
  • Microscopy, Confocal / instrumentation
  • Microscopy, Fluorescence, Multiphoton / instrumentation
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Photons
  • Rats
  • Secretory Vesicles / chemistry
  • Secretory Vesicles / drug effects
  • Secretory Vesicles / ultrastructure
  • Serotonergic Neurons / chemistry*
  • Serotonergic Neurons / ultrastructure
  • Serotonin / analysis*
  • Serotonin / pharmacology
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods
  • Ultraviolet Rays


  • Serotonin