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.

In this study, we developed a novel, compact, and efficient structured illumination microscopy (SIM) system, to our best knowledge. A binary hexagonal lattice pattern was designed and implemented on a digital micromirror device (DMD), resulting in a projection-based structured-light generation. By leveraging the combination of the high-speed switching capability of the DMD with a high-speed CMOS camera, the system can capture 1024×1024 pixels images at a 200 fps frame rate when provided with sufficient illumination power. The loading of the hexagonal lattice pattern reduces the number of images required for reconstruction to seven, and by utilizing the DMD modulating characteristics on the illumination path, there is no need to use bulky mechanical structures for phase shifting. We designed a compact system with 110mm×150mm×170mm dimensions that displayed a 1.61 resolution enhancement for fluorescent particle and biological sample imaging.