Abstract Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two-photon imaging is shown to enable a quantitative in situ analysis of such fiber–single-neuron coupling in the live brain of transgenic mice. Fiber-optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two-photon imaging. These results pave the way for fiber-optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber-optic single-neuron stimulation into the optical imaging framework.