Phase-multiplexed continuous Bessel droplet for high-contrast, gapless volumetric two-photon microscopy

Extended depth-of-focus (DOF) imaging using Bessel beams has advanced rapid volumetric two-photon fluorescence microscopy (2PFM) by enabling high-resolution imaging across large volumes. However, this technique faces challenges due to significant sidelobe excitation, which reduces image contrast and the signal-to-background ratio (SBR), particularly at high numerical apertures (NAs). Although Bessel droplets have been proposed to suppress sidelobes, they introduce axial discontinuities, leading to incomplete coverage of the sample volume. To achieve comprehensive volumetric imaging of biological specimens, secondary or multiple axial scans with displaced Bessel droplets are typically required, which inevitably increases acquisition time. In this work, we introduce the phase-multiplexed continuous Bessel droplet (PM-CBD), generated via a four-channel phase multiplexing approach, to address these limitations. The PM-CBD suppresses sidelobes effectively while maintaining the extended DOF and diffraction-free properties of Bessel beams. By eliminating the axial gaps present in Bessel droplet imaging, PM-CBD enables rapid, high-contrast, and complete volumetric imaging. Our results show that PM-CBD improves the SBR, and reduces primary sidelobes by approximately 40% compared to traditional Bessel beams. This technique provides a practical enhancement to existing 2PFM platforms, offering improved imaging performance for in vivo applications in neuroscience and developmental biology.