The temporal and spatial dynamics of neurotransmitter release are fundamental to understanding activity-dependent signaling between axons and other cells, including neurons, glia, and vascular cells. A microscopic imaging technique is described that enables studying release of the neurotransmitter ATP from axons in response to action potentials. The method combines imaging single-photons, intrinsic optical signal imaging, and high magnification time-lapse microcopy to enable investigations of action potential-induced ATP release together with cell morphology and activity-dependent axon swelling. ATP released from axons catalyzes a chemiluminescent reaction between luciferin and luciferase that generates single photons that can be imaged individually. In addition to vesicular release, ATP release through membrane channels activated by axon swelling was monitored simultaneously with intrinsic optical signals. Repeated emissions of photons were observed from localized 15 μm regions of axons, with a frequency distribution that differed from a normal distribution and from the frequency of emissions outside these localized regions.