On-line Optical Clearing Method For Large Tissues Imaging

Humans know very little about the brain.Understanding the mechanism of brain operation will help us overcome brain diseases and promote the development of artificial intelligence.The brain structure is basis for realizing various brain functions,and neurons as its basic unit form a network through various local and cross-brain connections to achieve specific brain functions.So deciphering the brain structure at single-cell level is fundamental to understand brain operating mechanism.Macaque,as a non-human primate,has a high similarity to human brain.It is irreplaceable in the study of advanced brain functions and mental diseases,compared with widely-used rodent animals.However,the volume of the macaque brain is about 200 times that of the mouse,and there is no effective method to image the whole brain of the macaque monkey with a single-cell resolution.In response to this problem,this thesis proposes a solution for imaging monkey brains: on-line optical clearing treatment combined with tissue sectioning and microscopic imaging.This thesis developed a new method of on-line optical clearing,which is to perform the clearing processing of macaque brain sample during sectioning.Improve the imaging efficiency of large-field imaging system and an efficient agarose embedding scheme is adopted.It converts the way of ultra-thin sectioning into a certain thickness vibrating sectioning,avoiding the requirement for large-area high-precision tissue sectioning.Imaging the macaque brain tissue with a single-cell resolution has been realized.This thesis compares the current method of optical clearing and chooses a watersoluble reagent based on passive immersion for the on-line clearing method.The author also establishes a rapid clearing penetration model and studies as well as compares the penetration characteristics of different saturated reagents for fast clearing.The fast clearing effects of sorbitol,sucrose,fructose,and glycerin had been analyzed.It had been found that fructose was significantly better than the others for fast clearing.Therefore,fructose had been selected as the main component of the fast-clearing reagent.Urea had also been added to the fructose solution to optimize the refractive index of the solution.Sample swelling due to the hydration reaction between urea and biological tissues offsets the sample shrinkage due to dehydration.The overall deformation is controlled by about 5%.The experiment further verified that the screened clearing reagent had no significant effect on fluorescent proteins brightness in the sample.The author employed agar-embedded mice brains to verify the effectiveness of the online optical clearing method.In this thesis,a customized whole brain imaging system consisting of a structured illumination microscope and a vibrating sectioning is used to realize the automatic fast acquisition of whole mouse brain dataset.Due to the change in the solution viscosity,the parameters of the vibrating sectioning were optimized,and the surface flatness of the samples had been controlled within 10 ?m.In order to correct the problem of data mismatch between adjacent layers caused by optical clearing,a method of marked cells as characteristic points to register optical clearing images is proposed,which realizes the accurate three-dimensional registration of on-line optical clearing data.The results of whole brain imaging mouse brain show that the on-line optical clearing method doesn't require clearing processing in the early stage,ultra-thin sectioning,and the system imaging efficiency is doubled.To apply this method to larger macaque brain samples,the reagent formulation of online optical clearing was further optimized.The long-term stability of the fast clearing reagent was studied and it was found that the oxidation reaction was one of the main reasons for the decreased clearing effect.It was found that adding 1-mercaptoglycerol could inhibit the oxidizing reaction of the reagent,which improved the long-term stability of the fast clearing solution for macaque monkey brain imaging.In addition,to overcome that the saturated solution crystallized during the long time imaging process for macaque brain,the solution concentration was reduced and promoting agent was added as the main component for imaging macaque brain block.Finally,a mixture of 30% fructose + 20% urea solution +5% propanediol + 0.5% 1-thioglycerol solution was selected as an improved fast clearing reagent,and the supporting imaging scheme in 3D large volume was optimized.This method is also compatible with on-line propidium iodide staining to obtain cytoarchitecture synchronously.Finally,it takes about 10 days to realize the 3D imaging of the macaque monkey brain block in the size of 30 × 30 × 20 mm3,and obtain the distribution and colocation cytoarchitecture of fluorescent labeled neurons.This method is expected to provide a new method for the study of whole-brain fine structure of large samples,such as macaques and even human brains.