Fluorescence of serotonin in the visible spectrum upon multiphotonic photoconversion

The vital molecule serotonin modulates the functioning of the nervous system. The chemical characteristics of serotonin provide multiple advantages for its study in living or fixed tissue. Serotonin has the capacity to emit fluorescence directly and indirectly through chemical intermediates in response to mono- and multiphoton excitation. However, the fluorescent emissions are multifactorial and their dependence on the concentration, excitation wavelength and laser intensity still need a comprehensive study. Here we studied the fluorescence of serotonin excited multiphotonically with near-infrared light. Experiments were conducted in a custom-made multiphoton microscope coupled to a monochromator and a photomultiplier that collected the emissions. We show that the responses of serotonin to multiphoton stimulation are highly non-linear. The well-known violet emission having a 340 nm peak was accompanied by two other emissions in the visible spectrum. The best excitor wavelength to produce both emissions was 700 nm. A green emission with a ∼ 500 nm peak was similar to a previously described fluorescence in response to longer excitation wavelengths. A new blue emission with a ∼ 405 nm peak was originated from the photoconversion of serotonin to a relatively stable product. Such a reaction could be reproduced by irradiation of serotonin with high laser power for 30 minutes. The absorbance of the new compound expanded from ∼ 315 to ∼ 360 nm. Excitation of the irradiated solution monophotonically with 350 nm or biphotonically with 700 nm similarly generated the 405 nm blue emission. Our data are presented quantitatively through the design of a single geometric chart that combines the intensity of each emission in response to the serotonin concentration, excitation wavelengths and laser intensity. The autofluorescence of serotonin in addition to the formation of the two compounds emitting in the visible spectrum provides diverse possibilities for the quantitative study of the dynamics of serotonin in living tissue.