Hydrogel Embedding Method For Three-dimensional High-resolution Imaging Of Macaque Brain

Due to the high genetic homology,macaques are the most closely related research animals to humans.Obtaining anatomical information of neurons and blood vessels in the macaque brain is essential in exploring brain functions.Optical imaging is an effective technology to obtain mesoscale information on brain tissue.However,the high scattering of light in biological tissues makes it difficult to directly image deep tissue continuously.Tissue embedding combined with mechanical sectioning could avoid high light scattering in the deep tissue to accomplish whole brain imaging.However,most of the current embedding methods are only suitable for small organs such as mouse brains.Since the volume of macaque brain is about 200 times that of mouse brain,and its structure is extremely complex,there are no reports on the method of embedding macaque brain.Therefore,it is essential to develop a new embedding method that allows preservation of anatomical information and fluorescent signals of macaque brains and are suitable for mechanical sectioning which could help overcome light scattering interference.My main work is summarized in the following passages.(1)A hydrogel embedding method that preserved the anatomical structure and fluorescent signals of biological samples.By comparing the stability and the degree of cross-linking of different hydrogels with biological tissues,I selected N-acryloyl glycinamide(NAGA)as the main embedding agent.X-ray tomography and confocal microscopy imaging results showed that the sample volume did not change significantly during 3 months of storage.In addition,the lengths and branch angles of axons and blood vessels stayed the same,which showed the fine anatomical structures were maintained.Furthermore,with the initiator VA-044 and optimized polymerization temperature,the fluorescence retention rate of GFP,YFP,and td Tomato were increased to over 200%respectively,and this method was also compatible with a variety of fluorescent probes.(2)A hydrogel embedding scheme suitable for three-dimensional continuous imaging of intact organs.To adjust the mechanical properties of the hydrogel,I introduced the crosslinking agent NN-methylene diacrylamide.The mechanical sectioning roughness was2?4?m after the adjustment,which met the requirements for continuous three-dimensional imaging.As a result,I obtained high-resolution information of neuronal and vascular structures in the whole mouse brain.In order to increase tissue transparency and achieved higher imaging speed,I established a new method named M-CUBIC(Machinable CUBIC),a combination of mechanical sectioning and tissue clearing.Through this method,the light transmittance of brain tissue was increased from 40%to 90%,the effective imaging depth was increased from 50?m to 150?m,and the imaging time was saved by about 1/2.(3)Hydrogel embedding and three-dimensional high resolution structure acquisition of macaque brains.To increase the degree of cross-linking between the sample and the embedding agent,I proposed an agarose-biological sample-hydrogel crosslinking method.I acquired the cytoarchitectonic and angioarchitecture information of the macaque brain with a spatial resolution of 0.32×0.32×10?m~3.I also reconstructed individual long-range projecting axons that crossed the whole hemisphere of macaque brains with a spatial resolution of 0.65×0.65×3?m~3 for the first time.It was found that axons of pyramidal tract neurons in layer 5b may projected to the central dorsal thalamus and formed clustered axon terminals.The PNAGA hydrogel embedding method developed in this thesis can obtain the fine structure information of the whole macaque brain,which proved that the method could stably maintain the fine structure and fluorescence signal for a long time.And the mechanical properties of embedded samples could be adjusted to meet the requirements of mechanical sectioning performance in three-dimensional imaging of large volume samples.Compared with the current embedding methods applied to mouse brain size samples,this method greatly expanded the three-dimensional high-resolution imaging to the whole macaque brain,providing an efficient scheme for mesoscopic brain mapping of non-human primates.