Space-Time Light-Sheet Microscopy

Abstract Light-sheet microscopy (LSM) has revolutionized bioimaging by delivering high-contrast volumetric resolution with minimal photodamage. Spatial wavefront shaping, used to generate lattice and Airy light-sheets, has been particularly effective in advancing LSM beyond the Rayleigh limit. Despite its broad adoption, most LSM implementations rely on rigid dual-objective geometries that complicate sample handling and impose a trade-off between imaging field of view (FoV) and axial resolution. Here, we introduce space-time light-sheet microscopy (ST-LSM), a single-objective strategy that exploits space-time (ST) correlations for the first time. ST-LSM goes beyond separate spatial or temporal modulation to jointly modulate the spatiotemporal spectral structure of a pulse. This uniquely enabled light-sheets with wavelength-scale thickness over millimeter-scale distances. When compared to state-of-the-art approaches, ST-LSM eliminates the dual-objective constraint, expands the sample-accessible volume by 25×, and increases the FoV by 10× without sacrificing sectioning resolution. We demonstrate the versatility of ST-LSM by using a single setup to image specimens across four orders of magnitude in size, from whole roots and developing embryos, down to mammalian cells with sub-cellular axial resolution. These results position ST-LSM as an accessible and high-performance optical microscopy platform at a variety of biological scales, by translating space-time wave-packet physics into a practical imaging modality. Competing Interest Statement The University of Idaho has filed a provisional patent application (63/805,828) regarding the ST-LSM method, in which JZ, HL, DNC, and AEV are co-inventors. The remaining authors declare no competing interests.