Light-sheet excitation-encoded volumetric spectroscopy for fast multiplexed imaging and quantitative physicochemical mapping in cells

Understanding how cellular organization relates to function requires imaging approaches that resolve structural and physicochemical information throughout three-dimensional volumes. Here we introduce light-sheet excitation spectroscopic microscopy (LS-ExSM), a method that captures full excitation spectra with ∼10 nm spectral resolution at each voxel for multiplexed imaging and quantitative spectroscopic readout. Excitation-wavelength encoding makes this spectroscopic readout compatible with single-objective wide-field light-sheet imaging while maintaining high spectral fidelity. Combined with sparse volumetric sampling and deep-learning-based reconstruction, LS-ExSM achieves high-speed volumetric imaging while preserving structural and spectral information. We demonstrate six-color volumetric imaging of live cells at near-second temporal resolution with minimal crosstalk, enabling visualization and quantitative analysis of dynamic organelle interactions in three dimensions. We further apply LS-ExSM to three-dimensional mapping of lipid-droplet polarity at near-diffraction-limited resolution. Together, these results establish excitation-encoded volumetric spectroscopy as a route to whole-cell imaging of subcellular organization and physicochemical state.