Supercritical-lens light-sheet fluorescence microscopy with sub-diffraction-limit axial resolution, enhanced FOV, and chip-scale illumination.

Light-sheet fluorescence microscopy (LSFM) is widely used in biological volumetric imaging. However, further breakthroughs in axial resolution or improvements in field of view (FOV) suffer from higher numerical aperture (NA) or bulky and complex illumination paths, which challenge the design of LSFM, as the detection and illumination paths have to be compactly arranged in a finite space for the orthogonal spatial configuration. Here, a supercritical lens (SCL) is designed for a sub-diffraction-limit quasi-non-diffracting light sheet, where sidelobes are suppressed to 7.6% without a window, and an SCL-based LSFM is proposed to address these challenges. In-vivo volumetric imaging of zebrafish is successfully performed by the SCL-based LSFM. Experimental results illustrate it not only achieves sub-diffraction-limit axial resolution, i.e., axial resolution higher thanthe theoretical limit determined by the illumination path's NA, but also exhibits an enhanced FOV with almost no divergence. Importantly, these improvements can be easily achieved by a chip-scale illumination arm, an SCL composed of 100-nm-thick Cr microstructure, and 300-µm-thick glass substrate, enabling a simple and highly compact LSFM system. This work, for the first time, demonstrates the potential application of SCL in three-dimensional biological imaging and offers a promising approach for the design of highly integrated, high-performance, and chip-based LSFMs in the future.