Populational and individual axon projections of thalamic neurons in rhesus monkeys [Image: Professor Bi Guoqiang and Professor Lau Pakming’s team]

A team led by Professor Bi Guoqiang and Professor Lau Pakming at the University of Science and Technology of China (USTC) of the Chinese Academy of Sciences (CAS) and at CAS’s Shenzhen Institute of Advanced Technology (SIAT), and their collaborators, have realized three-dimensional (3D) mapping of the entire brain of the macaque monkey at micron resolution. The achievement was 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 and three-dimensional microscopy with semiautomated reconstruction and tracing (SMART). The study was published in Nature Biotechnology.

Our brain is comprised of nearly a hundred billion nerve cells with delicate and complex connections between 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 monkey, although highly desired given their roles in modeling human diseases, has been hindered by a major technical challenge. The brain of a rhesus monkey, more than 200 times larger in volume than that of a mouse, is simply too big.

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

Schematic of VISoR2 [Image: Professor Bi Guoqiang and Professor Lau Pakming’s team]

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 images, allowing for unblurred imaging when the sample is in continuous motion. In this way, VISoR achieves more than ten times the speed of other 3D imaging methods for large tissue samples.

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

Their improved VISoR2 system allowed them to finish imaging 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.

The researchers also 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.

Professor David C. Van Essen from Washington University in St Louis commended 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 huge imaging data obtained with AI analysis, it may be possible to understand the fine 3D structure of the brain and body as well as how it changes 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 Professor 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 Professor Wang Xiaojing of New York University.

Source: University of Science and Technology of China,

Chinese Academy of Sciences