Abstract Long-term and high-spatiotemporal-resolution 3D imaging of living cells remains an unmet challenge for super-resolution microscopy, owing to the noticeable phototoxicity and limited scanning speed. While emerging light-field microscopy can mitigate this issue through threedimensionally capturing biological dynamics with merely single snapshot, it suffers from suboptimal resolution insufficient for resolving subcellular structures. Here we propose an adaptive light-field microscopy (VCD-LFM 2.0) with new optics-aware and adaptive-tuning deep learning strategies (VCD 2.0) capable for highly-generalizable light-field reconstruction of diverse subcellular dynamics. VCD-LFM 2.0 accomplishes isotropic spatial resolution down to 120 nm without compromising superior temporal resolution and low phototoxicity. With the development of a compact light-field add-on and an open-source reconstruction software, we realize rapid (up to 333 volumes per second), sustained (up to 60 hours) 3D super-resolution imaging of diverse intracellular dynamics simply on a retrofitted inverted microscope. With VCD-LFM 2.0 approach, we quantitatively analyze both the instantaneous processes of lysosome-mitochondrial interaction and long-term evolution of mitochondrial across two complete cell cycles.

Optics-aware super-resolution light-field microscopy for long-term volumetric imaging of dynamic intracellular processes at millisecond timescales