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Abstract Creating a high-resolution brain atlas in diverse species offers crucial insights into general principles underlying brain function and development. A volume electron microscopy approach to generate such neural maps has been gaining importance due to advances in imaging, data storage capabilities, and data analysis protocols. Sample preparation remains challenging and is a crucial step to accelerate the imaging and data processing pipeline. Here, we introduce several replicable methods for processing the brains of the gastropod mollusc,Berghia stephanieaefor volume electron microscopy. Although high-pressure freezing is the most reliable method, the depth of cryopreservation is a severe limitation for large tissue samples. We introduce a BROPA-based method using pyrogallol and methods to rapidly process samples that can save hours at the bench. This is the first report on sample preparation and imaging pipeline for volume electron microscopy in a gastropod mollusc, opening up the potential for connectomic analysis and comparisons with other phyla. 

Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies. 

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.