High-contrast volumetric imaging of synapses and microcirculations with Bessel-droplet two-photon fluorescence microscopy

Bessel foci used for two-photon fluorescence excitation have enabled high-speed volumetric imaging. At high numeric aperture, their imaging performances are compromised by substantial side-ring excitation and suffer from reduced image contrast. Here, we describe axially extended Bessel-droplet foci with suppressed side rings and more resistance to optical aberrations. Applying novel phase patterns to generate Bessel-droplet foci of variable NAs at high power throughput, we achieved continuous volume imaging by scanning them interferometrically along the axial direction. With Bessel-droplet foci, we demonstrated high-resolution volumetric imaging of synaptic anatomy and function as well as lymphatic circulations in the mouse brain in vivo.