利用二倍頻顯微術分析角膜中的 膠原蛋白結構與特性

Structural Observation of Cornea Collagen Organization by Second Harmonic Generation Microscopy

楊博安(Po-An Yang)

指導教授 ： 董成淵

國立臺灣大學/理學院/應用物理所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10925

摘要

近年來，雙光子光學顯微術時常被使用在生物影像的研究，非線性光學顯微術相較於一般的光學顯微術，它的優點是可以點激發，避開在聚焦點以外的樣本被激發，並且可以使用更高波長的光源激發生物樣本，可以減少對於生物樣本的破壞，以及加深掃描的深度，因此非線性光學顯微術非常適合用在三維生物組織的影像掃描中。
角膜是眼睛的最外層的組織，主要是由膠原蛋白所組成。當角膜內的膠原蛋白結構被破壞時，會嚴重影響人們的視力，甚至失明。想要製造出人造角膜並且用它來治癒因角膜受損而導致的疾病時，必須要有合適的分析方法去研究角膜的結構。在這篇論文中，我們使用二維傅立葉轉換分析角膜中的膠原蛋白排列。藉由二維傅立葉轉換分析雞和吳郭魚眼角膜中膠原蛋白的穿透式二倍頻訊號，我們發現從角膜表層到內層，膠原蛋白呈順時針方向旋轉，而且當在越靠近角膜內層時，旋轉的變化變得越慢。由於角膜中膠原蛋白的反射式二倍頻的訊號較為模糊，所以我們無法藉由二維傅立葉轉換分析得到膠原蛋白的角度，因此我們使用可解析角度之二倍頻分析角膜中的膠原蛋白的角度。可解析角度之二倍頻主要是藉由膠原蛋白的二倍頻強度會隨著入射光的極化角度與膠原蛋白的夾角改變而隨之變化以找出膠原蛋白的方向。實驗後我們發現可以藉由可解析角度之二倍頻分析角膜的反射式二倍頻訊號，找出角膜內的膠原蛋白的排列，這讓我們發現它未來可以被運用在觀察活體的角膜結構生成的可能性。

關鍵字

二倍頻 ； 膠原蛋白 ； 角膜

並列摘要

In recent years, two-photon excitation microscopy (TPEM) has been widely used in the biological and medical imaging. Due to the limited sub-femtoliter focal volume, images with high depth-discrimination can be obtained with TPEM. Limited focal volume also significantly reduces off-focal photo-damage. Furthermore, light at longer wavelength allows deeper image penetration into the tissue. These advantages enable non-linear optical microscopy be a popular imaging method in biological sciences.
Cornea, the outermost layer of the eye, is mainly composed of fibrotic collagen. Any distortion of cornea collagen orientation can result in significant visual deterioration. In order to understand how corneal collagen is organized and to fabricate the artificial cornea to cure corneal diseases, it is important to have a technique that is capable of analyzing the structure of the cornea. We first utilizing two dimensional fast fourier transform (2D-FFT) to obtain the structural characteristics of corneal collagen. By using 2D-FFT to analyze forward second harmonic generation (SHG) image of cornea along with depth, we find orientation of collagen fibrils in chicken and Tilapia cornea both rotated counterclockwise when observing from anterior to posterior portion of the cornea. Furthermore, the varying rate of collagen orientation decreases with increasing the depth of cornea. Due to 2D-FFT failing to distinguish homogeneous image such as backward SHG, we apply polarization sensitive second harmonic generation to analyze the cornea structure. By analyzing differences of second harmonic generation (SHG) at different angle between polarization and collagen fibril, the orientation of the collagen fibrils in the cornea can be deduced with pixel-resolution. We find that PSSHG can be used to analyze the backward SHG which suggesting the potential of using PSSHG to observe the corneal development in vivo.

並列關鍵字

Cornea ； Second Harmonic Generation ； collagen

參考文獻

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