適用于熒光蛋白標(biāo)記大樣本的塑性包埋方法研究
發(fā)布時(shí)間：2018-10-14 19:31
【摘要】：獲取高分辨率的小鼠全腦神經(jīng)元連接圖譜對(duì)認(rèn)識(shí)腦的信息處理機(jī)制、疾病致病機(jī)理和發(fā)育異常等具有重要意義。神經(jīng)系統(tǒng)中表達(dá)熒光蛋白的小鼠模型是研究腦的結(jié)構(gòu)和功能連接網(wǎng)絡(luò)的有力工具，但現(xiàn)有的成像技術(shù)尚無(wú)法在大范圍內(nèi)獲取熒光蛋白標(biāo)記神經(jīng)元的精細(xì)連接關(guān)系。近年發(fā)展起來(lái)的基于塑性包埋和超薄切片的顯微光學(xué)層析成像技術(shù)，典型代表如可實(shí)現(xiàn)亞微米分辨的顯微光學(xué)切片斷層成像系統(tǒng)，被認(rèn)為是目前最有效的對(duì)大體積樣本進(jìn)行微米甚至更高水平的精細(xì)結(jié)構(gòu)三維成像的方法之一。然而，這類(lèi)技術(shù)還難以應(yīng)用于熒光蛋白標(biāo)記的大樣本（比如完整的小鼠腦），最主要的原因是缺少可行的樣本制備方法。因此，本文旨在發(fā)展一種能適用于熒光蛋白標(biāo)記大樣本的塑性包埋方法，并將其應(yīng)用于小鼠全腦的精細(xì)連接結(jié)構(gòu)研究。 針對(duì)樹(shù)脂包埋的熒光保持率進(jìn)行了定量測(cè)試和優(yōu)化。本文提出了基于100μm厚的小鼠腦片和雙光子顯微鏡的熒光變化測(cè)量方法。定量研究了黃色熒光蛋白（YellowFluorescent Protein, YFP）經(jīng)六種樹(shù)脂分別包埋后的熒光強(qiáng)度變化，發(fā)現(xiàn)這些樹(shù)脂的熒光保持率從大到小依次為HPMA、Technovit8100、JB-4、GMA、Unicryl和LR White。在此基礎(chǔ)上，優(yōu)化了GMA的配方和使用方法，并首次使得YFP在GMA樹(shù)脂包埋后的熒光保持率提高了近一倍。 針對(duì)水環(huán)境下樹(shù)脂的切片性能進(jìn)行了研究。以顯微光學(xué)切片斷層成像系統(tǒng)為實(shí)驗(yàn)平臺(tái)，建立了評(píng)估樹(shù)脂在水環(huán)境下的切片性能的研究方法，，并用于多種樹(shù)脂固化塊的水中1μm切片性能研究，研究發(fā)現(xiàn)HPMA、GMA、Unicryl、LR White等樹(shù)脂可在長(zhǎng)時(shí)間水浸泡條件下進(jìn)行連續(xù)穩(wěn)定的1μm切片。 針對(duì)大樣本中樹(shù)脂的滲透性能進(jìn)行了研究。以小鼠全腦作為生物大樣本，建立了測(cè)試樹(shù)脂在大樣本中的滲透速度的研究方法，并用于評(píng)估不同樹(shù)脂的滲透速度，研究發(fā)現(xiàn)LR White、Unicryl、GMA在合適的條件下2~3天內(nèi)可充分滲透小鼠全腦，而HPMA滲透小鼠全腦需要2周以上的時(shí)間。 基于前述研究結(jié)果，建立了一套完整的適用于熒光蛋白標(biāo)記小鼠全腦的GMA樹(shù)脂包埋方法。利用該方法制備的Thy1-eYFP-H小鼠全腦樣品能夠在可實(shí)現(xiàn)熒光成像的顯微光學(xué)切片斷層成像系統(tǒng)中進(jìn)行連續(xù)穩(wěn)定的1μm厚度切片，同時(shí)實(shí)時(shí)采集YFP熒光圖像，最終可獲得體素1μm分辨率的小鼠全腦數(shù)據(jù)集。通過(guò)對(duì)該數(shù)據(jù)集進(jìn)行圖像預(yù)處理和三維重建，可顯示熒光蛋白標(biāo)記的神經(jīng)元在小鼠全腦三維空間中的長(zhǎng)程投射路徑。 隨著轉(zhuǎn)基因小鼠模型資源的不斷豐富，本文發(fā)展的塑性包埋方法有潛力在獲取哺乳動(dòng)物神經(jīng)網(wǎng)絡(luò)甚至是血管網(wǎng)絡(luò)中發(fā)揮重要作用。本文建立的塑性包埋方法還可以推廣應(yīng)用到多色熒光蛋白標(biāo)記組織以及其它動(dòng)物器官等等的樣品制備和高分辨率結(jié)構(gòu)數(shù)據(jù)獲取中。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R310
本文編號(hào)：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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R310
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Yasuaki Shibata,Tomoo Tsukazaki,Akira Yamaguchi;The Comparison of the Effects of Alcohol and Acetone on Green Fluorescent Protein Intensity[J];解剖科學(xué)進(jìn)展;2005年02期
本文編號(hào)：2271426
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