Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation

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Abstract
Tryptophan and serotonin were imaged with infrared illumination by three-photon excitation (3PE) of their native ultraviolet (UV) fluorescence. This technique, established by 3PE cross section measurements of tryptophan and the monoamines serotonin and dopamine, circumvents the limitations imposed by photodamage, scattering, and indiscriminate background encountered in other UV microscopies. Three-dimensionally resolved images are presented along with measurements of the serotonin concentration (∼50 mM) and content (up to ∼5 × 10 8 molecules) of individual secretory granules.