Off-axis compressed encoding microscopy for high-resolution, high-throughput, high-contrast volumetric imaging

High-throughput volumetric fluorescence microscopy requires depth-dependent information to be preserved while maintaining lateral resolution, optical-sectioning contrast, and acquisition speed. Here, we introduce off-axis compressed encoding microscopy (OCEM), a volumetric imaging method that encodes depth-dependent information directly into accelerated two-dimensional measurements. OCEM achieves physics-defined depth encoding: the illumination profile and off-axis multiline detection geometry generate detector-row-specific intensity fingerprints that vary with axial position. These fingerprints provide depth constraints directly in the acquired measurements, enabling a lightweight decoder to recover depth-resolved volumes with optical-sectioning contrast. OCEM combines 4× axial compression with 4× lateral acceleration, achieving a 16-fold increase in volumetric acquisition throughput based on camera exposure time. Because the encoding is implemented within the existing off-axis detection geometry, OCEM requires no additional beam splitters, cameras, or detection paths. We validate OCEM in whole-brain imaging and volumetric FISH, demonstrating preserved structural continuity, optical-sectioning contrast, and signal-localization consistency for large-scale quantitative biological imaging.