Monoamine neurotransmitters are implicated in mood, aggression, reward, and addiction pathways in the mammalian brain. However, visualizing them in live neurons with sub-micron resolution has remained a challenge. It is difficult to label them fluorescently, and their intrinsic ultraviolet fluorescence is difficult to access. Unlike serotonin which can be imaged with three-photon microscopy, dopamine presents a special challenge due to its shorter wavelength (∼300nm) emission. We now show that dopamine can be imaged with sub-micron resolution in live brain slices with a combination of a non-epifluorescent collection design, special optical elements, and two-photon excitation with a visible femtosecond laser. Substantia Nigra (SN) tissue sections from the rat show a group of cells much brighter than those observed in non-Nigral control regions. They contain hundreds of dopamine vesicles/vesicle clusters in the soma, akin to the serotonergic neurons from the Raphe imaged earlier. Drugs such as amphetamine are thought to cause non-exocytotic release of dopamine from the dopaminergic neurons but measuring such dynamics has been difficult. Our time-lapse imaging shows that 100 μM para-chloroamphetamine depletes the total dopamine content of SN neurons by about 25% over a time scale of fifteen minutes without substantial translocation of the vesicles. Imaging neurotransmitters in live cells also allows us to monitor the differentiation of stem-cells into serotonergic neurons over a period of many weeks. Interestingly, we observe that the ability to exocytose serotonergic vesicles arises several days after serotonin starts expressing in these cells. Our imaging techniques therefore provide a visual assay for a more functionally relevant maturation point for the differentiation of these cells, a fact with possible clinical significance for the treatment of various neurodegenerative and injury-related disorders.