Large Field of View and Isotropic Light Sheet Microscopy with Aberration‐Free Tunable Foci

Abstract Light‐sheet microscopy stands out as a powerful tool in biological imaging due to its exceptional performance in fluorescence imaging. However, achieving both high sectioning performance and a vast field of view (FOV) poses a fundamental challenge in conventional light‐sheet microscopy. The light‐sheet thickness is typically constrained to 1 µm for a wide FOV, potentially compromising resolution. To address this limitation, an axial scanning light‐sheet microscopy (ASLM) technique integrated with aberration‐free tunable foci to enable high‐NA excitation while maintaining a generous FOV, is introduced. The proposed scheme successfully achieves isotropic resolution of 280 nm in a 3D imaging system, encompassing a FOV of 80 × 80 µm and an impressive imaging speed of 80 ms per frame. These remarkable characteristics underscore the immense potential of ASLM for high‐ spatiotemporal resolution imaging.