Noninvasive rapid observation of volumetric calcium transients in human cortical organoids using light field microscopy with annular scanning

Volumetric calcium imaging in three-dimensional neural cultures requires high temporal throughput, cellular-level spatial discrimination, and low phototoxicity; however, conventional axial-scanning methods trade volumetric speed against cumulative excitation dose. Here, we present an optical-computational imaging platform that geometrically matches annular-scanning (AS) excitation to the light-field microscopy (LFM) reconstruction volume. By spatially redistributing excitation into a time-averaged oblique conical geometry, AS suppresses out-of-focus fluorescence and reduces local photon density while preserving single-shot volumetric detection. Post-reconstruction denoising further recovers signal fidelity near the shot-noise floor. Together, AS-LFM with denoising offers a several-fold signal-to-noise ratio improvement and sustained image contrast throughout the ballistic imaging regime. This optical-computational gain enables scan-free 354 × 354 × 72 μm3 volumetric reconstruction at 15 Hz and observation of over 60 spontaneously active neurons of GCaMP6f-expressing human cortical organoids at the excitation power reduced to 17.9 μW during continuous over 60 min recordings. The system resolved heterogeneous spontaneous activity, axially overlapping neurons with distinct temporal dynamics, and characterized diverse functional phenotypes, including tonic firing, bursting, and synchronized network events. Pharmacological validation using tetrodotoxin and 4-aminopyridine (4-AP) demonstrated sensitivity to physiologically relevant perturbations of suppression or enhancement of network activity, with 4-AP producing increases in event rates accompanied by marked alterations in functional connectivity patterns. These results establish excitation-engineered LFM as a low-phototoxic, scan-free volumetric detection strategy for sustained functional imaging of human three-dimensional neural organoid cultures.