Abstract

Long-term observation of subcellular dynamics in living organisms is limited by background fluorescence originating from tissue scattering or dense labeling. Existing confocal approaches face an inevitable tradeoff among parallelization, resolution and phototoxicity. Here we present confocal scanning light-field microscopy (csLFM), which integrates axially elongated line-confocal illumination with the rolling shutter in scanning light-field microscopy (sLFM). csLFM enables high-fidelity, high-speed, three-dimensional (3D) imaging at near-diffraction-limit resolution with both optical sectioning and low phototoxicity. By simultaneous 3D excitation and detection, the excitation intensity can be reduced below 1 mW mm⁻², with 15-fold higher signal-to-background ratio over sLFM. We imaged subcellular dynamics over 25,000 timeframes in optically challenging environments in different species, such as migrasome delivery in mouse spleen, retractosome generation in mouse liver and 3D voltage imaging in Drosophila. Moreover, csLFM facilitates high-fidelity, large-scale neural recording with reduced crosstalk, leading to high orientation selectivity to visual stimuli, similar to two-photon microscopy, which aids understanding of neural coding mechanisms.