Expanding horizons: a decade of synergy between light sheet and expansion microscopy

Abstract Over the past decade, biological imaging has been revolutionized by the convergence of two distinct technologies: expansion microscopy (ExM) and light sheet fluorescence microscopy (LSFM). ExM bypasses the diffraction limit of light by embedding biological specimens in a swellable polyelectrolyte hydrogel and physically expanding them, thereby decoding molecular structures. However, this process results in a massive increase in sample volume – scaling from a 64-fold volumetric increase (derived from a 4-fold linear expansion) up to a 1,000-fold volumetric increase (derived from a 10-fold linear expansion) – which renders traditional point-scanning microscopy prohibitively slow and phototoxic. LSFM, with its ability to illuminate samples with a thin sheet of light and detect fluorescence via wide-field cameras, offers the necessary speed and gentle imaging conditions to map these expanded volumes. This review synthesizes the evolution of “ExLSM” from 2014 to 2025, detailing the progression from early proof-of-concept studies to the development of lattice light-sheet integration and the recent breakthrough of Expansion-Assisted Selective Plane Illumination Microscopy (ExA-SPIM). It further examines the parallel innovations in “universal” hydrogel chemistry, spatial omics integration, and the petabyte-scale computational infrastructure now required to process the resulting data.