适用于荧光蛋白标记大样本的塑性包埋方法研究
发布时间：2018-10-14 19:31
【摘要】：获取高分辨率的小鼠全脑神经元连接图谱对认识脑的信息处理机制、疾病致病机理和发育异常等具有重要意义。神经系统中表达荧光蛋白的小鼠模型是研究脑的结构和功能连接网络的有力工具，但现有的成像技术尚无法在大范围内获取荧光蛋白标记神经元的精细连接关系。近年发展起来的基于塑性包埋和超薄切片的显微光学层析成像技术，典型代表如可实现亚微米分辨的显微光学切片断层成像系统，被认为是目前最有效的对大体积样本进行微米甚至更高水平的精细结构三维成像的方法之一。然而，这类技术还难以应用于荧光蛋白标记的大样本（比如完整的小鼠脑），最主要的原因是缺少可行的样本制备方法。因此，本文旨在发展一种能适用于荧光蛋白标记大样本的塑性包埋方法，并将其应用于小鼠全脑的精细连接结构研究。 针对树脂包埋的荧光保持率进行了定量测试和优化。本文提出了基于100μm厚的小鼠脑片和双光子显微镜的荧光变化测量方法。定量研究了黄色荧光蛋白（YellowFluorescent Protein, YFP）经六种树脂分别包埋后的荧光强度变化，发现这些树脂的荧光保持率从大到小依次为HPMA、Technovit8100、JB-4、GMA、Unicryl和LR White。在此基础上，优化了GMA的配方和使用方法，并首次使得YFP在GMA树脂包埋后的荧光保持率提高了近一倍。 针对水环境下树脂的切片性能进行了研究。以显微光学切片断层成像系统为实验平台，建立了评估树脂在水环境下的切片性能的研究方法，，并用于多种树脂固化块的水中1μm切片性能研究，研究发现HPMA、GMA、Unicryl、LR White等树脂可在长时间水浸泡条件下进行连续稳定的1μm切片。 针对大样本中树脂的渗透性能进行了研究。以小鼠全脑作为生物大样本，建立了测试树脂在大样本中的渗透速度的研究方法，并用于评估不同树脂的渗透速度，研究发现LR White、Unicryl、GMA在合适的条件下2~3天内可充分渗透小鼠全脑，而HPMA渗透小鼠全脑需要2周以上的时间。 基于前述研究结果，建立了一套完整的适用于荧光蛋白标记小鼠全脑的GMA树脂包埋方法。利用该方法制备的Thy1-eYFP-H小鼠全脑样品能够在可实现荧光成像的显微光学切片断层成像系统中进行连续稳定的1μm厚度切片，同时实时采集YFP荧光图像，最终可获得体素1μm分辨率的小鼠全脑数据集。通过对该数据集进行图像预处理和三维重建，可显示荧光蛋白标记的神经元在小鼠全脑三维空间中的长程投射路径。 随着转基因小鼠模型资源的不断丰富，本文发展的塑性包埋方法有潜力在获取哺乳动物神经网络甚至是血管网络中发挥重要作用。本文建立的塑性包埋方法还可以推广应用到多色荧光蛋白标记组织以及其它动物器官等等的样品制备和高分辨率结构数据获取中。
[Abstract]:The acquisition of high-resolution neuronal connectivity map of mouse whole brain is of great significance in understanding the mechanism of information processing disease pathogenesis and abnormal development of the brain. The mouse model of fluorescent protein expression in nervous system is a powerful tool to study the structure and function of brain junction network, but the existing imaging technology can not obtain the fine connection of fluorescent protein labeled neurons in a large range. Recently developed microscopic optical tomography technology based on plastic embedding and ultra-thin slice, such as micro-optical slice tomography system, which can realize sub-micron resolution, has been developed in recent years. It is considered to be one of the most effective methods for fine structure 3D imaging of large volume samples in micrometer or higher level. However, it is difficult to apply this technique to large samples labeled with fluorescent proteins (such as intact mouse brains), mainly due to the lack of feasible sample preparation methods. Therefore, this paper aims to develop a plastic embedding method which can be used to label large samples of fluorescent proteins and apply it to the study of fine junctional structure of the whole brain of mice. The fluorescence retention rate of resin embedding was quantitatively tested and optimized. In this paper, a fluorescence measurement method based on 100 渭 m thick mouse brain slice and two photon microscope is proposed. The fluorescence intensity changes of yellow fluorescent protein (YellowFluorescent Protein, YFP) were quantitatively studied after embedding with six kinds of resins. It was found that the fluorescence retention rates of these resins were HPMA,Technovit8100,JB-4,GMA,Unicryl and LR White. from large to small. On this basis, the formulation and application method of GMA were optimized, and the fluorescence retention of YFP in GMA resin was increased nearly double for the first time. The properties of resin slicing in water environment were studied. Based on the microoptical slice tomography system, a method to evaluate the slice properties of resin in water was established and used to study the properties of 1 渭 m slice in water of various resin solidified blocks. It was found that HPMA,GMA,Unicryl,LR White and other resins could be used for continuous and stable 1 渭 m slicing under the condition of long time water immersion. The permeability of resin in large sample was studied. Using the whole brain of mice as a biological sample, a method for measuring the osmotic rate of resin in large sample was established, and the method was used to evaluate the osmotic rate of different resins. It was found that LR White,Unicryl,GMA could fully penetrate the whole brain of mice within 2 days under suitable conditions. It takes more than 2 weeks for HPMA to penetrate the whole brain of mice. Based on the above results, a complete GMA resin embedding method applied to the whole brain of mice labeled with fluorescent protein was established. The whole brain samples of Thy1-eYFP-H mice prepared by this method can be continuously sliced with 1 渭 m thickness in a microoptical slice tomographic system which can realize fluorescence imaging, and real-time YFP fluorescence images can be acquired at the same time. Finally, the mouse global brain dataset with 1 渭 m resolution of voxel was obtained. By image preprocessing and 3D reconstruction, the long range projection pathway of fluorescent protein-labeled neurons in the three-dimensional space of mouse brain can be demonstrated. With the abundance of transgenic mouse model resources, the plastic embedding method developed in this paper has the potential to play an important role in the acquisition of mammalian neural networks and even vascular networks. The plastic embedding method developed in this paper can also be applied to the preparation of polychromatic fluorescent protein labeled tissues and other animal organs and the acquisition of high-resolution structural data.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R310
本文编号：2271426
[Abstract]:The acquisition of high-resolution neuronal connectivity map of mouse whole brain is of great significance in understanding the mechanism of information processing disease pathogenesis and abnormal development of the brain. The mouse model of fluorescent protein expression in nervous system is a powerful tool to study the structure and function of brain junction network, but the existing imaging technology can not obtain the fine connection of fluorescent protein labeled neurons in a large range. Recently developed microscopic optical tomography technology based on plastic embedding and ultra-thin slice, such as micro-optical slice tomography system, which can realize sub-micron resolution, has been developed in recent years. It is considered to be one of the most effective methods for fine structure 3D imaging of large volume samples in micrometer or higher level. However, it is difficult to apply this technique to large samples labeled with fluorescent proteins (such as intact mouse brains), mainly due to the lack of feasible sample preparation methods. Therefore, this paper aims to develop a plastic embedding method which can be used to label large samples of fluorescent proteins and apply it to the study of fine junctional structure of the whole brain of mice. The fluorescence retention rate of resin embedding was quantitatively tested and optimized. In this paper, a fluorescence measurement method based on 100 渭 m thick mouse brain slice and two photon microscope is proposed. The fluorescence intensity changes of yellow fluorescent protein (YellowFluorescent Protein, YFP) were quantitatively studied after embedding with six kinds of resins. It was found that the fluorescence retention rates of these resins were HPMA,Technovit8100,JB-4,GMA,Unicryl and LR White. from large to small. On this basis, the formulation and application method of GMA were optimized, and the fluorescence retention of YFP in GMA resin was increased nearly double for the first time. The properties of resin slicing in water environment were studied. Based on the microoptical slice tomography system, a method to evaluate the slice properties of resin in water was established and used to study the properties of 1 渭 m slice in water of various resin solidified blocks. It was found that HPMA,GMA,Unicryl,LR White and other resins could be used for continuous and stable 1 渭 m slicing under the condition of long time water immersion. The permeability of resin in large sample was studied. Using the whole brain of mice as a biological sample, a method for measuring the osmotic rate of resin in large sample was established, and the method was used to evaluate the osmotic rate of different resins. It was found that LR White,Unicryl,GMA could fully penetrate the whole brain of mice within 2 days under suitable conditions. It takes more than 2 weeks for HPMA to penetrate the whole brain of mice. Based on the above results, a complete GMA resin embedding method applied to the whole brain of mice labeled with fluorescent protein was established. The whole brain samples of Thy1-eYFP-H mice prepared by this method can be continuously sliced with 1 渭 m thickness in a microoptical slice tomographic system which can realize fluorescence imaging, and real-time YFP fluorescence images can be acquired at the same time. Finally, the mouse global brain dataset with 1 渭 m resolution of voxel was obtained. By image preprocessing and 3D reconstruction, the long range projection pathway of fluorescent protein-labeled neurons in the three-dimensional space of mouse brain can be demonstrated. With the abundance of transgenic mouse model resources, the plastic embedding method developed in this paper has the potential to play an important role in the acquisition of mammalian neural networks and even vascular networks. The plastic embedding method developed in this paper can also be applied to the preparation of polychromatic fluorescent protein labeled tissues and other animal organs and the acquisition of high-resolution structural data.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R310
【参考文献】
相关期刊论文 前1条
1 Yasuaki Shibata,Tomoo Tsukazaki,Akira Yamaguchi;The Comparison of the Effects of Alcohol and Acetone on Green Fluorescent Protein Intensity[J];解剖科学进展;2005年02期
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