<p>Precise control of the firing of an individual neuron in vivo is a key technology to neuroscience. The laser-induced ion channel opening makes it possible to depolarize neurons and trigger the firing. In this study, we present a noninvasive, opsin-free photostimulation method for activating an individual neuron within the primary visual cortex (V1). This activation is achieved through the transient local scanning of a tightly focused femtosecond laser on the soma of the target neuron that opens the store-operated calcium channels by multiphoton excitation, induces Ca²⁺ influx, and depolarizes the neurons to trigger action potentials (APs) firing. In the absence of any visual stimuli, the isolated activation of an individual neuron in a cortical ensemble in layer 2/3 of V1 is sufficient to elicit visually guided specific behaviors in awake mice, without co-activating other neurons in the ensemble. Remarkably, the disruption of a single neuron within the ensemble temporarily paralyzes the entire ensemble and suspends behavioral responses to visual stimuli. However, the ensemble rapidly recovers its responsiveness and function. In general, this opsin-free photostimulation method activates targeted individual ensemble neurons in visual cortex of awake mice enabling firing APs and eliciting behaviors.</p>