共軛焦顯微鏡是一種成熟的商用系統，常用於獲取次微米等級解析度的螢光生物圖像。然而，一般高數值孔徑物鏡的水平視野仍然限於數十微米乘以數十微米的範圍，而其垂直軸的視野穿透深度也因生物組織中的散射效應被限制。為了構建一個完整的果蠅神經元資料庫，在本研究中，整合組織澄清、軟組織切片、電動平移台和共軛焦顯像等技術建立了一個自動化體積共軛焦成像系統，並成功地以自動掃描取像的方式,在保持樣本不收縮變形的情況下，取得3毫米 x 2毫米 x 1毫米大小的完整果蠅樣本三維影像，內含神經元, 肌肉和外殼層等訊息，以此影像可初步建立果蠅全身神經影像標準模型，並將果蠅行為學研究中觀察到的特殊行為果蠅的神經影像嵌入此模型中，藉此研究大腦與身體之間的神經元聯結。
此外，本系統的技術應用不僅限於神經學研究，亦能夠拓展至其他動物軟組織大體積樣本取像，本研究中以此系統提供細胞核、細胞質甚至是免疫染色標記的三維生物組織病理學圖像，搭配深度學習人工智能演算法開發，可分析出病理組織中目標特徵區域，並進一步計算特徵區域的三維參數曲線。本技術於癌症組織檢體中，相較於傳統二維病理影像所提供之平面資訊更為全面，並且貼近組織原始樣貌型態，可完整呈現具有特異性的三維組織樣貌，為下一世代精準醫療提供更準確的診斷輔助資訊。

Confocal microscopy is a well-developed, commercially available system to acquire fluorescence biological images in sub-micron level resolution. However, the restricked field of view of high N.A. objective lenses and the penetration depth caused by scattering effects in turbid tissue, limited the application of this system. In this research, an automated large volume 3D imaging system was built up via integrated tissue clearing, three axis microtome, and confocal imaging techniques together, expand the image capability of confocal microscopy to millimeter scale. An automated scanning-sectioning loop was established with this system and have successfully acquire a whole Drosophila sample 3D image including neuron, muscle and cuticle channels which is 3 mm × 2 mm × 1 mm in size without shrinkage or distortion. This result demonstrated a preliminary representative whole Drosophila neuron model, which having the potential to build a comprehensive Drosophila neuron circuit. This model could be futher linked to Drosophila behavior experiment by expression neuron circuit pattern registration for studying the connectome between brain and body.
Furthermore, the application of this system not only limit into neuron research, but also capable to providing three-dimensional bio-tissue histopathology image with the nucleus, cytoplasmic, or even specific proteins labeling, these images could be implemented with artificial intelligence analysis software for next-generation precision diagnostic application.

摘要
Abstract
Chapter 1 – 緒論--------------------3
Chapter 2 - 系統設計與開發------------7
2.1 系統設計理念----------------------7
2.2 共軛焦顯微鏡系統------------------13
2.3 高速光學掃描系統------------------17
2.4 三維振盪切片機--------------------23
2.5 浸潤式組織澄清試劑-----------------29
Chapter 3 – 控制與分析軟體------------34
3.1 控制軟體與使用者介面設計------------34
3.2 分析軟體--------------------------36
3.2.1腫瘤分類模型開發-------------------38
3.2.2 腫瘤分割模型開發------------------45
Chapter 4 - 樣本製備-------------------50
4.1 製備材料簡介------------------------50
4.2 樣本製備流程------------------------52
Chapter 5 – 系統應用成果與討論-----------64
5.1 全果蠅體神經網路應用-----------------64
5.2三維組織病理應用----------------------71
Chapter 6 – 結論-----------------------78
參考文獻-------------------------------81
附錄-----------------------------------86

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