Tunable waist control in axially swept light-sheet microscopy for optimized signal and axial resolution

Ever since its inception, axially swept light-sheet microscopy (ASLM) has demonstrated its diverse imaging capability, particularly in the context of large, cleared specimens. Yet current implementations face fundamental limitations in optimizing imaging performance for heterogeneous samples. Although significant advances have been made in ASLM development, obtaining sufficient signal while maintaining optimal axial resolution remains a persistent challenge. Moreover, current ASLM implementations use fixed illumination numerical apertures (NAs) or lossy mechanical slits, preventing dynamic light-sheet waist control, thereby restricting multiscale imaging flexibility. Here, we present an ASLM-based optical method that enhances sample signal detection without the need to increase laser power while providing flexibility in axial resolution control ranging from 500 nm to 1.6 μm. Our approach enables flexible and lossless control of light-sheet thickness in the sample space to achieve variable axial resolution within the specified range. This tunable beam shaping capability addresses the fundamental limitations of current ASLM systems by providing researchers with the ability to optimize imaging parameters by bridging the critical gap between resolution and signal in ASLM-based microscopy. We demonstrate the utility of our approach by imaging fluorescent beads and various cleared-tissue samples.