High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural mechanisms, and can be a powerful tool for retinal disease diagnosis and treatment outcome evaluation. Fast intrinsic optical signals (IOSs), which have the time courses comparable to that of electrophysiological activities in the retina, hold the promise for high resolution imaging of retinal neural activities. However, application of fast IOS imaging has been hindered by the contamination of slow, high magnitude optical responses associated with transient hemodynamic and metabolic changes. In this paper we demonstrate the feasibility of separating fast retinal IOSs from slow optical responses by combining flicker stimulation and dynamic (temporal) differential image processing. A near infrared flood-illumination microscope equipped with a high-speed (1000 Hz) digital camera was used to conduct concurrent optical imaging and ERG measurement of isolated frog retinas. High spatiotemporal resolution imaging revealed that fast IOSs could follow flicker frequency up to at least 6 Hz. Comparable time courses of fast IOSs and ERG kinetics provide evidence that fast IOSs are originated from stimulus activated retinal neurons. © 2010 Optical Society of America.