Abstract: Optogenetic systems and methods for probing a specimen using spatio-temporally modulated illumination light are disclosed. A method may include generating illumination light, the illumination light including a plurality of illumination protocols temporally sampled and interleaved with one another at a time-division-multiplexed (TDM) sampling rate, each illumination protocol being for illuminating a respective region of interest (ROI) of a plurality of ROIs of the specimen. The illumination light may include either activation or excitation light, or both. The method may also include applying a spatio-temporal modulation to the illumination light and directing the resulting modulated illumination light onto the specimen.