Multiphoton imaging of abnormal serotonin oxidation products
Using MPM it is also possible to detect aberrant indoleamines produced in vivo by abnormal oxidation chemistry of serotonin (5-HT) and related compounds. Normally, excess 5-HT is enzymatically converted to compounds such as 5-hydroxyindoleacetic acid (5-HIAA), one of several stable oxidized forms of indoleamine. However under conditions of oxidative stress aberrant products of indoleamine oxidation can form. These abnormal oxidation products have been implicated in the etiology of several neurodegenerative diseases and the neurodegeneration arising from amphetamine usage. The primary aberrant oxidation products of 5-HT -- dihydroxytrypamines and dione forms of tryptamine -- undergo rapid dimerization resulting in fluorescent compounds that are thought to be the source of the toxicity. The absorption and emission spectra of 5-HT are shown along with spectra of the dimer and trimer emission peaks created by overnight air-oxidation at room temperature (a). The dimer fluorescence is separable from the "normal" indoleamine emission at 350 nm and the spatial coincidence of these two emissions allows for the measurement of the oxidative state of the indoleamine pools within cells. Using 3PE of 5-HT and simultaneous 2PE of the dimers formed by abnormal oxidation, it is possible to image changes in the oxidative state of granule-residing indoleamines in a mucosal mast cell line. As an example a change in the ratio of blue to UV emission is induced by oxidative stress resulting from the application of small amounts of peroxide (b).