Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.