PySight: Plug and play photon counting for fast continuous volumetric intravital microscopy

Imaging increasingly large neuronal populations at high rates pushed multi-photon microscopy into the photondeprived regime. We present PySight, an add-on hardware and software solution tailored for photon-deprived imaging conditions. PySight more than triples the measured median amplitude of neuronal calcium transients in awake mice and facilitates single-trial intravital voltage imaging in fruit flies. Its unique data streaming architecture allowed us to image a fruit fly’s brain olfactory response over 234 μm × 600 μm × 330 μm at 73 volumes per second, while retaining over 200 times lower data rates than those of a conventional data acquisition system with comparable voxel sizes (1.2 μm × 1.2 μm × 2.2 μm). PySight requires no electronics expertise or custom synchronization boards, and its open-source software is extensible to any imaging method based on single-pixel (bucket) detectors. PySight offers an optimal data acquisition scheme for ever increasing imaging volumes of turbid living tissue.