The intricate workings of the brain are embedded within complex networks of neuronal activityspanning a wide range of spatial and temporal scales. To investigate these dynamic processes, this dissertation presents the development of a novel, real-time closed-loop framework—the SpatioTemporal Illumination Microscope (STIMscope). This platform enables simultaneous large field-of-view imaging (up to 7.8 × 6 mm²) and patterned optical stimulation, all within an open-source, customizable, and cost-effective design. A real-time synchronized control system— supported by custom firmware and graphical user interface (GUI)—facilitates seamless communication between components, ensuring precise control and coordination. Extensive numerical simulations, optical modeling, and experimental measurements are provided to validate the proposed approach. Collaborative research demonstrates a broad spectrum of STIMscope applications, showcasing its potential to deliver unprecedented neural imaging and neuromodulation in head-fixed mice as well as in cell and tissue cultures. In addition, this dissertation introduces the Multiwell STIMscope, designed for high-throughput imaging and patterned stimulation in 96-well plates. By integrating the base large field-of-view capabilities of the STIMscope with engineered miniaturized lens stacks, the Multiwell STIMscope enables single-cell imaging and neuromodulation within a compact, high-density format. Validation results underscore its performance and highlight its potential for applications in high-throughput drug discovery and screening.