Two-beam Interference Microscopy: high-resolution multi-scale light-sheet imaging from subcellular structures to whole organs

Abstract Despite offering rapid and gentle 3D imaging of biological samples, the revolution by light-sheet fluorescence microscopy faces challenges due to optical constraints and limited geometry flexibility, hindering its widespread application. Here we present Two-beam Interference Microscopy (TIM), which sweeps the interference pattern of two symmetric laser sheets to achieve horizontal plane illumination and orthogonal wide-field detection for the first time using a single objective. The geometry-unconstrained TIM yields adaptive optical sectioning of samples with various sizes and mounting approaches. It enables high-speed (over 150 megavoxels/s), adaptive-resolution (~200 nm to ~2500 nm), nanoscale-to-mesoscale 3D imaging ranging from single organelles within a live cell (×100), to a whole mouse brain containing tens of millions of neurons (×4 tile stitching). TIM exhibits remarkably high versatility while achieving superior spatiotemporal resolution. With feedback-controlled imaging strategies, intelligent TIM demonstrates in situ trans-scale visualization from entire brain slices to single dendritic spines, and efficient event-triggered imaging of CAR-T/tumor cells’ cytotoxic interactions.