Stretching the boundaries: Expansion microscopy a game changer in super-resolution imaging.

Super-resolution microscopy (SRM) has transformed the study of cellular structures, enabling imaging beyond the diffraction limit. Yet, the need for costly instrumentation has limited its accessibility. Expansion Microscopy microscopy (ExM), pioneered by Ed Boyden's laboratory, offers an alternative by physically enlarging samples embedded within a swellable hydrogel. This simple principle makes nanoscale resolution achievable with conventional fluorescence microscopy. Since its introduction in 2015, ExM has rapidly diversified. Iterative ExM (iExM) increases resolution through repetitive expansion, chemical gel innovation enables single-step 10- to 20-fold expansion, and hybrid strategies combining ExM and SRM techniques have pushed resolution below 15 nm. ExM has now been applied to diverse biological models but its adaptation to complex plant tissues poses unique challenges due to their rigid cell walls. Recent advances in the field of plant science have started to address these obstacles, opening access to nanoscale imaging of plant cellular structures such as plasmodesmata and the mitotic spindle. In this review, we trace the development of ExM from its pioneering stages to current refinements, discuss methodological advances and hybrid approaches, examine technical limitations, and highlight emerging applications across biological models, with a particular focus on recent progress and future perspectives in plant biology.