Dense, Synapse-level Reconstruction of Mammalian Brain Tissue with Light Microscopy
Brain tissue comprises an extremely complex arrangement of cells that together make up the information processing
network enabling brain function. The connectivity of neurons underlies the unparalleled
capabilities of our mind. Thus, mapping brain structure, which underlies brain function, has become a
central focus in neuroscience.
Electron microscopy provides extremely high resolution and comprehensive visualization of brain
structure but requires correlative workflows to access molecular information. Light microscopy holds
tremendous potential to analyze the ultrastructure of brain tissue together with its molecular makeup.
However, conventional light microscopy (LM) has limited resolution (~ 200 nm laterally and ~ 1000 nm
axially), far too coarse to distinguish neuronal structures in comprehensively labelled tissue and to
precisely locate specific molecular players within sub-micrometer-sized structures, such as synapses.
We have developed an optical imaging approach based on high-fidelity hydrogel expansion to visualize
even the finest of neuronal structures, including axons and dendritic spines, when paired with
comprehensive structural labeling and imaged with diffraction-limited, high-speed confocal microscopy,
unveiling neuronal structure together with molecular information. This enables deep-learning based,
dense segmentation of neuronal structures and determining neuronal connectivity at single-synapse
resolution, which we showcase in mouse cortex and hippocampus. Our technology will help shed light on
brain structure, connectivity and molecular composition in a readily adoptable manner.