The team led by Prof. BI Guoqiang and Prof. LAU Pakming at University of Science and Technology of China (USTC) of the Chinese Academy of Sciences (CAS) and at Shenzhen Institute of Advanced Technology of CAS, and the collaborators, realized three-dimensional (3D) mapping of the entire brain of the macaque monkey at micron resolution based on an updated version of their recently developed high-throughput 3D fluorescence imaging technique VISoR, and an efficient pipeline combining serial sectioning and clearing, three-dimensional microscopy with semiautomated reconstruction and tracing (SMART). This study was published in Nature Biotechnology. 

Our brain comprises nearly a hundred billion nerve cells with delicate and complex connections among them. To fully understand how the brain functions, it is essential to have a high-resolution map showing how the nerve cells are organized and connected within the brain.

At present, it usually takes days to complete 3D imaging of the whole brain of a mouse at micron resolution using state-of-the-art techniques. However, high-resolution brain mapping for nonhuman primates such as the rhesus monkeyalthough highly desired given their roles in modeling human diseases, has been hindered by a major technical challenge. The brain of a rhesus monkey is too big, more than 200 times larger in volume than that of a mouse.

To overcome this challenge, the researchers developed a high-throughput 3D fluorescence imaging technique, Volumetric Imaging with Synchronous on-the-fly-scan and Readout (VISoR).

Compared with commonly used 3D optical imaging techniques, VISoR eliminates the time loss caused by moving and pausing while switching fields of view to obtain 2D image series, allowing for imaging without blur when the sample is in continuous motion. In this way, VISoR achieves very high speed, which is more than ten times faster than other 3D imaging methods for large tissue samples. 

In addition to the challenge of imaging throughput, difficulty of imaging monkey brains also arises from the complicated cortical folding structures and low tissue transparency. The researchers first sectioned the isolated brain into 0.3-mm slices and developed reagents to make it thoroughly transparent. 

With their improved VISoR2 system, they finished imaging of a whole macaque monkey brain in 100 hours at a resolution of 1×1×2.5 microns. The total volume of raw image data acquired from two macaque brains exceeded 1 PB.

Besides, the researchers developed efficient algorithms and software to realize automated 3D imaging reconstruction and semi-automated long-distance tracking of individual neuronal axon fibers. Their initial observations revealed previously unknown characteristics of axonal fiber projection and surprising patterns of fiber turning and routing in the cortical folds.

Prof. David C. Van Essen from Washington University in St Louis commented this work as a “technical tour de force that marks a stunning advance in our ability to map long-distance connectivity accurately and efficiently throughout the entire brain of the macaque monkey”. He said that besides the technical achievement, their exciting discovery may have profound implications for understanding brain morphogenesis and the principle of 'wiring length minimization'.

The application of VISoR may be extended to the imaging of other tissues and organs, including samples from clinical pathology. It is anticipated that by combining the obtained huge imaging data with AI analysis, this technique may help to understand the fine 3D structure of the brain and body as well as how they change in various disease conditions, thus facilitating medical diagnostics and drug developments.

“Hopefully, this technology will be further improved for broader and larger scale applications, to make important contributions to the mapping and understanding of primate and eventually the human brain,” said Prof. DUAN Shumin from Zhejiang University.

“Brain connectome at the mesoscopic level is important but so far limited to rodents. This work demonstrates a powerful method that enables researchers to dissect mesoscopic connectome of monkeys at one micron resolution, in four days. It represents a tour de force in this rapidly moving field,” said Prof. WANG Xiaojing from the New York University.

Populational and individual axon projections of thalamic neurons in rhesus monkeys (Image by Prof. BI Guoqiang and Prof. LAU Pakming's team)