- 學位論文
活體倍頻顯微術:長時間脊索動物胚胎發育之觀測
In vivo harmonic generation microscopy:Long term observations of vertebrate embryos development
摘要
在這篇論文中，我探討倍頻顯微術在胚胎發生學及胚胎基因學中所扮演的角色。我們實驗室之前已建立一套光學倍頻顯微鏡，並使用二倍頻和三倍頻來作為影像模態。我使用倍頻顯微術長時間活體觀察基因阻斷斑馬魚，發現某些特定的基因不止在神經發育過程中的神經系統表現，而是在胚胎發育初期就對胚胎造成非常大的影響。由此可證明倍頻顯微術確是一個理想觀察基因表現的工具。在結合一個工作在於生物穿透較深波段的近紅外光飛秒雷射光源和一個高數值孔徑的物鏡後，倍頻掃瞄顯微術可以提供超過一公厘的穿透深度及次微米的空間解析度，並在生物樣本中有著較少的光破壞及光毒性現象，因此可以拿來觀察許多生物體內的器官及胞器，並重組老鼠胚胎三維影像。很明顯的倍頻顯微術可以用來篩選老鼠甚至是人的胚胎。利用二倍頻和三倍頻的影像模態，倍頻顯微術不需要以侵入式的方式例如染劑染色或螢光蛋白標記就可在活體生物取得充足的影像資訊來幫助在生物學上的研究。 之前我們實驗室已成功的使用倍頻顯微術觀察活體內神經纖維的發育，但是我們並沒有去探討基因的功能。結合基因阻斷的技術，我可以三維的觀察特定基因在胚胎內的影響。我使用倍頻顯微術觀察zARNT2A 和 HIF 1
並列摘要
In this thesis, I discuss the role optical harmonic generation microscopy plays in morphant technology and embryogenesis of embryology. A previous optical harmonic generation microscopy system is built based on a modified optical scanning microscope and a Cr:forsterite laser. Second harmonic generation (SHG) and third harmonic generation (THG) have been used as imaging modalities. I present the in vivo long-term observation of morphant type zebrafish using HGM from the very beginning of embryogenesis, thus identify that the gene not only functions after nerve fiber formation in the nervous system, but up to the gastrula period of the embryo. With the continuous observation of the same morphant type embryo, optical harmonic generation microscopy is demonstrated as an excellent least invasive imaging tool for revealing gene expression in embryos. Combined with the near-infrared femtosecond source and a high numerical aperture (NA) objective, optical harmonic generation microscopy can offer >1-mm penetration depth and sub-micrometer spatial resolution in live biological samples with much-reduced photodamage and phototoxicity, and therefore it can reveal many organs and organelles in various vertebrate embryos and construct the whole 3D structure mouse embryo. Obviously HGM can be used to select mouse-even human- embryos as well. Combining SHG and THG imaging modalities, optical harmonic generation microscopy can provide biological critical information without invasive treatment such as dye staining or fluorescence protein labeling and contribute a better spatial resolution in embryology studies. Previously, our lab had successfully demonstrated the nerve fiber development through harmonic generation microscopy, but we cannot identify any gene function. With the aid of morphant technique, specific genes blocked by morpholinos have become a powerful tool in developmental biology studies. Based on a high-order harmonic generation microscope, images with high 3-dimensional (3D) resolution can be obtained due to its nonlinear nature. We have used higher-harmonic generation microscopy based on the femtosceond Cr:forsterite laser with the aid of zARNT2A and HIF 1
參考文獻
延伸閱讀
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