A simple Bessel module with tunable focal depth and constant resolution for commercial two-photon microscope

Abstract

The volumetric imaging of two-photon microscopy expands the focal depth and improves the throughput, which has unparalleled superiority for three-dimension samples, especially in neuroscience. However, emerging in volumetric imaging is still largely customized, which limits the integration with commercial two-photon systems. Here, we analyzed the key parameters that modulate the focal depth and lateral resolution of polarized annular imaging and proposed a volumetric imaging module that can be directly integrated into commercial two-photon systems using conventional optical elements. This design incorporates the beam diameter adjustment settings of commercial two-photon systems, allowing flexibility to adjust the depth of focus while maintaining the same lateral resolution. Further, the depth range and lateral resolution of the design were verified, and the imaging throughput was demonstrated by an increase in the number of imaging neurons in the awake mouse cerebral cortex.