Large-scale voltage imaging in the brain using targeted illumination

Recent improvements in genetically encoded voltage indicators enabled high precision imaging of single neurons action potentials and subthreshold membrane voltage dynamics in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool to record activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy prone to background signal contamination. We implemented a simple, low cost, targeted illumination strategy based on a digital micromirror device (DMD) to restrict illumination to the cells of interest to improve background rejection, and quantified optical voltage signal improvement in neurons expressing the fully genetically encoded voltage indicator SomArchon. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brains of awake mice. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over several minutes in the brain. Thus, the DMD-based targeted illumination strategy described here offers a simple solution for high-speed voltage imaging analysis of large scale network at the millisecond time scale with single cell resolution in the brains of behaving animals.