We propose a quasi-confocal microscopy autofocus system incorporating an electrically tunable lens (ETL) to achieve differential detection. The ETL changes its focal length to collect differential curves at speeds <300 Hz, allowing selective locking onto desired focal layers and high-speed differential operations close to the locked focal plane. By segmenting the system's pupil, the interference between the outgoing and incoming near-infrared beams is avoided, thereby greatly improving the signal-to-noise ratio. This ultra-sensitive system, with a focus drift accuracy better than 1/22 focal depth (∼20 nm @100× objective), provides a new, to the best of our knowledge, implementation pathway to meet the requirements of various microscopy techniques.
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