Chemical Sectioning Fluorescence Tomography: High-Throughput, High-Contrast, Multicolor, Whole-Brain Imaging at Subcellular Resolution

A thorough neuroanatomical study of the brain architecture is crucial for our understanding of its cellular compositions, connections and working mechanisms. However, the fine and multiscale features of neuron structures make it challenging for the microscopic imaging, as one cannot have both high-contrast, and high-throughput at the same time in such imaging. Here, we proposed the concept of chemical sectioning fluorescence tomography (CSFT) to solve this problem. Based on the switching characteristics of the fluorescent proteins (FPs), we introduced the chemical sectioning (CS) effect into the block-surface microscopy, turn off all FP molecules by chemical methods, and only light those in the top section as thin as submicron for imaging, so as to achieve high-throughput, high-contrast imaging without background. We developed a three-color wide-field fluorescence micro-optical sectioning tomography (fMOST) system. We proved that CSFT method was compatible with resin-embedded multicolor FPs and could be feasibly used in whole-brain imaging on diverse genetically or virally labeled long-projecting neurons and interneurons to the systematic reconstruction degree. We demonstrated mouse whole-brain imaging at the subcellular resolution, as well as the power for quantitative acquisition of synaptic connection-related pyramidal dendritic spines and axon boutons on the brain-wide scale.at the complete single-neuron level. We believe that CSFT method would greatly facilitate our understanding of the brain-wide neuron networks.