Study On A Plastic-embedding Method For FP Expressed Large Samples

High-resolution and brain-wide neural network is of particular importance to theunderstanding of the information processing mechanisms, disease pathogenesis anddevelopment of the brain. Currently, mouse model expressing fluorescent proteins (XFPs)in nervous system is a powerful tool in studying the structural and functional connectionsof the brain, but techniques for imaging the fine connections of XFPs-labeled neurons inlarge scale of the brain are lacked. Three-dimensional imaging method based onplastic-embedding and ultra-thin sectioning, among which the Micro-Optical SectioningTomography (MOST) is a representative, has been considered as one of the mosteffective techniques to acquire fine structure of a large sample at micron or even higherlevel. However, because of the limitations of sample preparation method, this kind ofimaging technique cannot be applied to the XFPs-labeled large sample. The purpose ofthis paper is to develop a plastic-embedding method suitable for XFPs-labeled largesample, which will be applied to obtain the fine connections of the whole mouse brain.The fluorescence preservation of different resins was tested and optimized. Based onthe100μm-thick brain sections and a two-photon microscope, we proposed a protocol formeasuring fluorescence change of YFP during resin embedding, and found that thefluorescence preservation of six resins is HPMA>Technovit8100>JB-4>GMA>Unicryl>LR White. In addition, we optimized the formulations and usage of GMA, bywhich the fluorescence preservation of YFP was nearly doubled.The sectioning performance of resins under water environment was studied. By usingMOST system as the experimental platform, we established a protocol for evaluating thesectioning performance of resins immersed in water. And this protocol was used to studythe1μm sectioning quality of six resins, which found that four of them, including HPMA,GMA, Unicryl and LR White, are suitable for long-term1μm sectioning under waterenvironment. The penetration ability of resins was studied. By using the whole mouse brain as alarge sample, we established a protocol for testing the penetrating speed of resins. Theresults showed that the LR White, Unicryl and GMA can fully penetrate into a wholemouse brain in2~3days, while HPMA spent more than two weeks.Finally, a plastic-embedding method, which is suitable for XFPs-labeled wholemouse brains, was developed based on the optimized GMA formulation. According to thismethod, a Thy1-eYFP-H mouse brain was prepared and then serial1μm sectioned andimaged with fluorescence MOST, by which a data set of the whole mouse brain wasobtained at1μm voxel resolution. Furthermore, by image pre-processing andreconstruction of the data set, the long-distance projection of a single YFP-labeled neuronwas demonstrated at the whole mouse brain scale.In future, with the development of mouse models, our novel plastic-embeddingmethod is potential to play an important role inreconstructing ofthe neuronal network ofmammals, as well as their vasculature. This method can be also applied to the samplepreparation and high-resolution data acquisition of multi XFPs-labeledtissues and otheranimal organs.