Imaging three-dimensional brain organoid architecture from meso- to nanoscale across development.

Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.