Full field-of-view hexagonal lattice structured illumination microscopy based on the phase shift of electro-optic modulators.

High throughput has become an important research direction in the field of super-resolution (SR) microscopy, especially in improving the capability of dynamic observations. In this study, we present a hexagonal lattice structured illumination microscopy (hexSIM) system characterized by a large field of view (FOV), rapid imaging speed, and high power efficiency. Our approach employs spatial light interference to generate a two-dimensional hexagonal SIM pattern, and utilizes electro-optical modulators for high-speed phase shifting. This design enables the achievement of a 210-µm diameter SIM illumination FOV when using a 100×/1.49 objective lens, capturing 2048 × 2048 pixel images at an impressive 98 frames per second (fps) single frame rate. Notably, this method attains a near 100% full field-of-view and power efficiency, with the speed limited only by the camera's capabilities. Our hexSIM demonstrates a substantial 1.73-fold improvement in spatial resolution and necessitates only seven phase-shift images, thus enhancing the imaging speed compared to conventional 2D-SIM.

DMD-based compact SIM system with hexagonal-lattice-structured illumination.

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 110m m×150m m×170m m dimensions that displayed a 1.61 resolution enhancement for fluorescent particle and biological sample imaging.

High-accuracy differential autofocus system with an electrically tunable lens.

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.