二倍頻(SHG)是一種適用於觀察生物組織中各向異性結構之三維分布的技術(如肌腱、心血管與真皮層等)，而這些部位皆富含I型膠原蛋白(Type-I Collagen)，此種物質中之非中心對稱結構與雙折射(Birefringence)特性，能使入射光產生強烈的二倍頻訊號，故此技術適用於醫療臨床端做為初期非侵入式的疾病診斷。但由於入射光源容易於生物組織內產生散射，進而導致在組織內的穿透深度降低，此缺點便侷限了二倍頻顯微術應用於厚的生物組織之研究領域。S. Rehn等人於2011年提出的文獻中，分別以程式模擬線性、橢圓以及圓偏振光，並傳播於一易散射物質。三種偏振光以圓偏振在物質內的穿透深度為最深，其次為橢圓偏振，穿透深度最淺的為線偏振，不過，由於此結果僅以程式模擬而獲得，並無法明確證實，故此結論仍須採用光路實驗佐證。
為此，本研究以波長範圍為808~810nm之飛秒鈦藍寶石雷射作為入射光激發源，並定義三種偏振光(線性偏振光、橢圓偏振光、圓偏振光)作為入射光；樣本採用不同厚度之豬的肌腱(30、60、100μm)作為薄樣本，採用豬的真皮層作為厚的樣本(300、600、700μm)；由於肌腱與皮膚皆為易散射樣本，因而，當一良好定義的偏振光在散射樣本中傳播時，會導致入射光的偏振型態改變，並降低在樣本內穿透的深度，故本研究透過量測入射光在通過厚、薄樣本以及無樣本條件下之偏振光強度比例得知，線偏振光的衰減程度最為明顯(可達-99%)，其次為橢圓偏振(可達-75%)，衰減最少的為圓偏振(可達55%)。同時，經本研究計算結果可得知，在薄樣本的環境下(厚度低於100μm)，三種偏振光的穿透深度皆相近，而在厚樣本的環境下(厚度大於600μm)，圓偏振的穿透深度為最深(約150μm)，橢圓偏振為第二(約140μm)，線偏振最淺(約120μm)。並證實可以透過改變入射光的偏振狀態進而提升在厚的易散射樣本中的穿透程度。

關鍵字: 二倍頻顯微術、非中心對稱、雙折射特性、偏振光、穿透深度

Second-harmonic generation (SHG) microscopy is a very useful technique for investigating the three-dimensional (3D) organization of anisotropic biological tissues, such as dermis, tendon and blood vessels. These are mainly composed of type-I collagen, which is structurally birefringent and of non-centrosymmetric structure, which can induce strong SHG signal. Therefore, this approach is suitable for clinical study as a non-invasive prediagnosis. However, large number of scattering events occurs between incident light and scattering particles in the tissue, hence it reduces the penetration depth of tissue and limits the application of SHG.
It has been reported that the degree of polarization of circular polarized light maintains initial polarization state for deeper penetration depth than the linearly or elliptically polarized light, which has been stimulated with a polarization-sensitive Moten Carlo model; however, this work was only obtained by programing simulation. Therefore, related estimations need to be confirmed by optical microscopy experiments.
In this work, we used a Ti:sapphire laser, providing the wavelength at around 810 nm to define three kinds of polarizations of incident beam, which are circular, elliptical and linear polarization. The forward SHG signal was obtained by PMT from pig’s tendon (30, 60, 100μm) and pig’s leather tissue (300, 600 and 700μm).
It is confirmed that the longest to shortest penetration depth is in the sequence of circular (150μm), elliptical (143μm) and linear (121μm) polarization when penetrating into thick sample. The percentage of polarization deterioration of circular, elliptical and linear polarization in different thick sample: the change of linear polarization is the most significant (-99%), second is elliptical polarization (-75%), the change in circular polarization is 55% that is the lowest.
Keyword: SHG, birefringent, non-central symmetric, polarized light, penetration depth

論文審定書 i
誌 謝 ii
摘 要 iii
Abstract iv
目 錄 v
圖目錄 vi
表目錄 ix
第一章 緒論 1
1.1前言 1
1.2研究動機與目的 2
1.3論文架構 4
第二章 研究理論 5
2.1 非線性光學 5
2.2 散射理論 6
2.3 消偏效應 10
2.4 偏振光穿透深度探討 12
2.5 二倍頻圓二色性(SHG-Circular Dichroism) 17
第三章 研究方式 19
3.1 研究方法 19
3.2 實驗操作步驟 28
第四章 結果與討論 38
4.1 三種偏振光之穿透深度差異 38
4.2偏振光受組織結構影響的偏振破壞程度 52
4.3二倍頻圓二色性分析結果 60
4.4雙折射特性對於穿透深度影響 64
第五章 結論與未來展望 66
5.1結論 66
5.2未來展望 68
參考文獻 69

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