High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level

Abstract

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

“…Finally, we embedded the IF labeled large-volume samples with SBB to lower background fluorescence [26]. We acquired 3D high-resolution images of large biological samples using the fMOST system [27].…”

“…The specimens were placed in a sealed drying vacuum oven at 50 °C for 8 hours. For the 1-mm-thick brain tissue block, we added 0.2% Sudan black B (SBB) [25,26] to the Lowicryl HM20 resin for the purpose of lowering the background fluorescence. We used a gelatin capsule (Electron Microscopy Sciences, cat.…”

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

“…With high programmable precision (100 nm) and feedback accuracy (20 nm) of the 3D precision motorized stage and rigidity of plastic embedding, the location error between two imaging processes was lot less than diffraction limit. Strip within same layer and frames from adjacent layer are registered naturally [4,26]. 200 nm fluorescent beads were used to measure spatial resolution of this system.…”

“…In brain science research, combining mechanical sectioning with regular imaging methods, researchers could obtain high resolution images in large volume [1][2][3][4][5]. However, limited by optics principle, the axial resolution of these methods, which mostly above 1 micron, would not be equally high as lateral resolution.…”

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

“…Thus, the strong out-of-focus interference in wide-field imaging is avoided via chemical reactivation of surface layer fluorescence. Combining the fluorescent proteins labeling, resin embedding and fluorescence micro-optical sectioning tomography (fMOST) [4,5], much work was done to visualize the neuronal morphology and neural circuits at brain-wide scale [6][7][8][9].…”