本研究主要探討我們研究團隊所發展的免疫染色結合同步加速器X-射線光源技術於果蠅的應用，透過這個技術，我們可以省略繁雜的解剖程序，保持果蠅頭部的完整性而得到特定的神經結構影像。我們主要聚焦在描繪和長期記憶相關的神經迴路，追蹤所有與這些記憶細胞相關的連結，進而繪製出完整的三維神經網路圖譜。繪製這些和記憶相關的神經結構圖譜有助於我們對突觸連接與神經形態的認識，並了解其功能以及在組織中所扮演的角色。我們使用國家同步輻射中心X-射線高強度且準直的光源，並藉由改進樣品的製備流程，以酵素金相學染色的高靈敏度特性，檢測核醣核酸序列以及其他特定的結合靶蛋白，並透過辣根過氧化物酶與金屬基質在特定基因表達上的化學反應，還原出金屬奈米粒子沉澱。我們根據實驗所得的藥劑條件、浸泡時間、酸鹼值以及添加劑建立一套新的染色流程，在無需進行大範圍的解剖下，達成奈米等級的解析目標。本研究提供了具備高度穩定性與可再現性的染色方法，以及果蠅嗅覺系統基因表達的X-射線原位重組影像，可為未來的神經科學研究進行更寬廣的發展與應用。

Three-dimensional reconstruction of neuronal wirings in the brain, which consist of cellular projections and synaptic connections, helps us understand the operation of nervous system in normal and disease individuals. Here, we show that synchrotron X-ray tomography enables us to image metal labeled fine structures within the whole Drosophila heads without complicated dissection. Enzyme Metallography (EnzMet) is a high sensitivity biological labeling and staining method for the detection of DNA, RNA and proteins. We developed a protocol using EnzMet − a peroxidase reducing a metal substrate to give enhanced staining – to label in situ gene expressions within the whole Drosophila head. We tested separate conditions to optimize EnzMet reaction, including measuring performance, reaction time, pH value, and additives at several levels. Imaging the metal labeled samples with the brilliance and highly collimated synchrotron X-ray, we have successfully reconstructed gene expression patterns and neuropilar structures in the whole Drosophila head at single neuron resolution. Comparing to the classical fluorescence confocal microscopy, the synchrotron X-ray imaging provides improved z-resolution and high-speed reconstruction of fine structures within intact large tissues, a feature critical for 3D reconstruction of connectomes in big brains.

Abstract in Chinese………………………………………………………………i
Abstract in English………………………………………………………………ii Acknowledgements………………………………………………………………iii
Table of Contents……………………………………………………………………iv
Chapter 1: Introduction…………………………………………………………1
Chapter 2: Materials and Methods………………………………………………4
2.1 Drosophila Husbandry………………………………………………………4
2.2 Drosophila Transgenesis……………………………………………………4
2.3 Tissue Fixation & Staining……………………………………………………4
2.4 Synchrotron X-ray Tomography & Imaging…………………………………5
2.5 Image Reconstruction……………………………………………………5
Chapter 3: Results………………………………………………………………6
3.1 Sample preparation and staining testing…………………………………6
3.1.1 Use of metals to identify peroxide reductases in tissues…………6
3.1.2 The influence of time on enzyme activity………………………………6
3.1.3 Reagent optimization for catalytic property and deposition efficiency…7
3.1.4 The pH values of the phenolic solution in the detection of AgNPs…8
3.1.5 Improvement of penetration by providing a decrease in cohesion……8
3.1.6 The polymer application in gold toning process………………………9
3.2 Accelerated X-ray Observation of Neurons (AXON) image processing…10
3.2.1 The X-ray phase imaging of nervous pathways………………………10
3.2.2 Synchrotron radiation-based tomographic reconstruction…………11
Chapter 4: Discussion……………………………………………………………12
List of Figures……………………………………………………………………16
References………………………………………………………………………26
Appendix………………………………………………………………………33

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