Video-rate volumetric functional imaging of the brain at synaptic resolution

Neurons and neural networks often extend hundreds to thousands of micrometers in three dimensions. To capture all the calcium transients associated with their activity, we need volume imaging methods with sub-second temporal resolution. Such speed is challenging for conventional two-photon laser scanning microscopy (2PLSM) to achieve, because of its dependence on serial focal scanning in 3D and the limited brightness of indicators. Here we present an optical module that can be easily integrated into standard 2PLSMs to generate an axially elongated Bessel focus. Scanning the Bessel focus in 2D turned frame rate into volume rate and enabled video-rate volumetric imaging. Using Bessel foci designed to maintain lateral resolution that resolves synapses in sparsely labeled brains in vivo, we demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice, and ferrets in vivo.