STIMscope: centimeter-scale all-optical imaging and patterned optogenetic manipulation at single-cell resolution

Linking observation to intervention at cellular resolution makes it possible to move from measuring network activity to testing the contribution of defined neurons or ensembles within the same preparation. All-optical probing provides this capability by combining fluorescence-based readout with targeted optogenetic manipulation. Yet the platforms that deliver this capability remain complex, expensive, and difficult to maintain, requiring specialized expertise that has confined them to a small number of laboratories. They also typically provide fields of view too limited for studies of large, distributed neuronal populations. We address these constraints with the Spatiotemporal Illumination Microscope (STIMscope), a one-photon benchtop platform that integrates large-aperture tandem optics with a small-pixel back-illuminated CMOS sensor, a digital micromirror device for patterned illumination, and a GPU-based processing unit coordinated by a microcontroller for hardware-level synchronization. Ray-tracing simulations and point-spread-function measurements confirm cellular-scale resolution, with imaging lateral FWHM of 5.6 μm at the field center and 5.8 μm at the edge, and excitation lateral FWHM of 5.8 μm at the center and 6.2 μm at the edge, supporting fields of view as large as 14 mm × 11 mm in the demagnified configuration. The accompanying Closed-loop ready Real-time Imaging and Stimulation Pipeline (CRISPI) provides GPU-accelerated calibrated mask projection (26.3 ms latency), online ROI trace extraction, and modular ZeroMQ-based control, with a measured imaging-to-stimulation loop benchmark of 91.6 ms. We validate STIMscope in fixed mouse brain tissue, live iPSC-derived human neuronal cultures, and ex vivo organotypic slices of mouse auditory cortex. In organotypic slices, we show that both static and spatiotemporal stimulus identity can be decoded from population activity, revealing reservoir-like population dynamics, and that this decodability remains stable in the same neuronal population over hours. We further show that post-stimulus activity retains information about recent stimuli for several seconds, consistent with short-term memory dynamics. With a bill of materials under $5,000 USD and all mechanical designs, firmware, and software released open-source, STIMscope makes all-optical neuroscience experiments a routine capability accessible to laboratories without specialized optical engineering expertise.