Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain. However, optical microscopy offer insufficient resolution to reveal subcellular details and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction there is a methodological gulf between electron-microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light-microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mmscale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.One Sentence Summary: Combined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes. 4 Main Text:Staring deep into the sky from a mountaintop in the American Southwest on a moonless night instills an emotional appreciation of the vastness of space, where the Milky Way galaxy, 60 billion times more massive than the Sun (1), represents but one of an estimated two trillion galaxies in the observable universe (2). Yet both our emotions and our intellect are made possible by the human brain, a 1.5 kg organ that, despite its small size, is no less complex and remarkable. There, over 80 billion neurons (3) connect through ~7,000 synapses each in a network of immense combinatoric complexity. Collectively, humanity creates a truly vast network containing more synapses than there are stars in the observable universe. Understanding the human mind is perhaps the most audacious undertaking in science today.Further underscoring this challenge is the knowledge that neural structures span a size continuum over seven orders of magnitude in extent, and are comprised of over 10,000 distinct protein types (4) collectively essential to build and maintain neural networks. For well over 100 years microscopy has played a central role in revealing this complexity (5,6). Electron microscopy (EM) has long been able to image down to the level of individual ion channels and synaptic vesicles (7), and today can extend this level of detail across the ~0.03 mm 3 volume of the brain of the fruit fly Drosophila melanogaster (8,9). However, EM creates a grayscale image where the segmentation of specific subcellular components or the tracing of the complete arborization of specific neurons remains challenging, and where specific proteins can rarely be unambiguously identified. Optical microscopy combined with immunofluorescence, fluorescent proteins, or fluorescence in situ hybridization (FISH) enables high sensitivity imaging of specific protein expression patterns in brain tissue (10, 11), brain-wide tracing of sparse neural subsets in flies (12, 5 13) and mice (14), and in situ identifi...

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