50 Hz volumetric functional imaging with continuously adjustable depth of focus

Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), which turns frame rate into volume rate by extending the excitation focus in axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the focal length. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 µm depth in larval zebrafish at 50 Hz volume rate with adjustable imaging thickness.