Axially sparse volumetric illumination-based light-field microscopy overcoming reconstruction limitations on z-ambiguity and near-NFP degradation

Light-field microscopy (LFM) captures volumetric fluorescence in a single exposure but is constrained by the spatial–angular sampling trade-off of a detector with a limited pixel count, leading to two major reconstruction limitations: z-ambiguity, which results in low and depth-dependent axial resolution, and severe reconstruction degradation near the nominal focal plane (NFP). Here, we present modulated light-sheet light-field microscopy (MoLiS-LFM), which acquires multiple z-sparse light-field images per volume using mutually independent, z-modulated light-sheet excitation. This approach effectively guarantees sparsity in the reconstructed volume and thereby improves axial resolving and localization capabilities. In addition, piezo-driven objective displacement shifts the NFP relative to the stationary specimen for a complementary MoLiS-LFM acquisition, leveraging LFM’s high-fidelity off-focus reconstruction to reduce near-NFP degradation. Fluorescent-bead experiments demonstrate improved axial resolving and localization capabilities, and whole-body C. elegans muscle imaging shows the recovery of structures near the NFP. To demonstrate the feasibility of MoLiS-LFM for in vivo imaging, we applied it to whole-brain imaging in larval zebrafish and achieved cellular-scale volumetric imaging over a 550 × 550 × 200~ μ m 3 volume at 9.06 volumes per second.