High pixel throughput voltage imaging based on repetitive optical selective exposure (ROSE)

Biological fluorescence imaging is constrained by a trade-off between field of view (FoV) and acquisition rate. This limitation is particularly evident with scientific complementary metal oxide semiconductor (sCMOS) cameras, where high frame rates force the maximum FoV into a narrow, high aspect ratio rectangle. Moreover, the rolling shutter used in high-speed imaging produces non-uniform exposure across the FoV, introducing artifacts when capturing rapid dynamics such as fast-moving objects or fluctuating fluorescence signals. These issues are especially detrimental to voltage imaging, a key technique for studying nervous system dynamics. Here, we introduce ROSE-based high-speed imaging (ROSE-HSI), a method that increases pixel throughput sixfold (when compared with a square FoV) by selectively exposing two cameras. ROSE-HSI not only overcomes the sampling rate limitations of a single camera but also eliminates the temporal inaccuracies caused by the rolling shutter. Our technique has enabled simultaneous kilohertz voltage imaging over a -pixel area, capturing distinct neuronal waveforms and synaptic-mediated signal propagation with high fidelity.