PySight: plug and play photon counting for fast intravital microscopy

Abstract

Imaging increasingly large neuronal populations at high rates pushed multi-photon microscopy into the photon-deprived regime. We present PySight, an add-on hardware and software solution tailored for photon-deprived imaging conditions. PySight more than triples the 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 olfactory response over 234×600×330µm 3 at 73 volumes per second, outperforming top-tier imaging setups while retaining over 200 times lower data rates. 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.

3567114.pdf

“…This protocol guides the prospective user through the necessary steps towards laser scanning microscopy with a MCS6A multiscaler. For your convenience, we provide customized settings files tailored for planar and volumetric two-photon microscopy in Code 1 [1].…”

“…This value, as well as most others, can be iteratively improved until a satisfactory result is achieved for a given acquisition system. The full explanation on the usage of PySight and its parameters can be found in the documentation of the Python package [1].…”