Continuous subcellular resolution three-dimensional imaging on intact macaque brain.

To decipher the organizational logic of complex brain circuits, it is important to chart long-distance pathways while preserving micron-level accuracy of local network. However, mapping the neuronal projections with individual-axon resolution in the large and complex primate brain is still challenging. Herein, we describe a highly efficient pipeline for three-dimensional mapping of the entire macaque brain with subcellular resolution. The pipeline includes a novel poly-N-acryloyl glycinamide (PNAGA)-based embedding method for long-term structure and fluorescence preservation, high-resolution and rapid whole-brain optical imaging, and image post-processing. The cytoarchitectonic information of the entire macaque brain was acquired with a voxel size of 0.32 µm × 0.32 µm × 10 µm, showing its anatomical structure with cell distribution, density, and shape. Furthermore, thanks to viral labeling, individual long-distance projection axons from the frontal cortex were for the first time reconstructed across the entire brain hemisphere with a voxel size of 0.65 µm × 0.65 µm × 3 µm. Our results show that individual cortical axons originating from the prefrontal cortex simultaneously target multiple brain regions, including the visual cortex, striatum, thalamus, and midbrain. This pipeline provides an efficient method for cellular and circuitry investigation of the whole macaque brain with individual-axon resolution, and can shed light on brain function and disorders.

DeepBrainSeg: Automated Brain Region Segmentation for Micro-Optical Images With a Convolutional Neural Network.

The segmentation of brain region contours in three dimensions is critical for the analysis of different brain structures, and advanced approaches are emerging continuously within the field of neurosciences. With the development of high-resolution micro-optical imaging, whole-brain images can be acquired at the cellular level. However, brain regions in microscopic images are aggregated by discrete neurons with blurry boundaries, the complex and variable features of brain regions make it challenging to accurately segment brain regions. Manual segmentation is a reliable method, but is unrealistic to apply on a large scale. Here, we propose an automated brain region segmentation framework, DeepBrainSeg, which is inspired by the principle of manual segmentation. DeepBrainSeg incorporates three feature levels to learn local and contextual features in different receptive fields through a dual-pathway convolutional neural network (CNN), and to provide global features of localization by image registration and domain-condition constraints. Validated on biological datasets, DeepBrainSeg can not only effectively segment brain-wide regions with high accuracy (Dice ratio > 0.9), but can also be applied to various types of datasets and to datasets with noises. It has the potential to automatically locate information in the brain space on the large scale.

A Robust Image Registration Interface for Large Volume Brain Atlas.

Accurately mapping brain structures in three-dimensions is critical for an in-depth understanding of brain functions. Using the brain atlas as a hub, mapping detected datasets into a standard brain space enables efficient use of various datasets. However, because of the heterogeneous and nonuniform brain structure characteristics at the cellular level introduced by recently developed high-resolution whole-brain microscopy techniques, it is difficult to apply a single standard to robust registration of various large-volume datasets. In this study, we propose a robust Brain Spatial Mapping Interface (BrainsMapi) to address the registration of large-volume datasets by introducing extracted anatomically invariant regional features and a large-volume data transformation method. By performing validation on model data and biological images, BrainsMapi achieves accurate registration on intramodal, individual, and multimodality datasets and can also complete the registration of large-volume datasets (approximately 20 TB) within 1 day. In addition, it can register and integrate unregistered vectorized datasets into a common brain space. BrainsMapi will facilitate the comparison, reuse and integration of a variety of brain datasets.

On-line optical clearing method for whole-brain imaging in mice.

The combination of optical clearing with light microscopy has a number of applications in the whole-brain imaging of mice. However, the initial processing time of optical clearing is time consuming, and the protocol is complicated. We propose a novel method based on on-line optical clearing. Agarose-embedded mouse brain was immersed in the optical clearing reagent, and clearing of the brain was achieved ~100 µm beneath the sample surface. After imaging, the cleared layer was removed, thereby allowing layer-by-layer clearing and imaging. No pre-immersion was required, and we demonstrated that on-line optical clearing can reduce the whole-brain imaging time by half.