利用二倍頻顯微術探討生物組織中膠原蛋白 結構與成分之特性

Structural Characterization of Collagen in Tissues by Second Harmonic Generation Microscopy

薛秋美(Chiu-Mei Hsueh)

指導教授 ： 董成淵

國立臺灣大學/理學院/物理研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00842

摘要

非線性光學顯微術近年來常被選取並應用在各種生物影像之研究;此項技術的優點包含光切片的功能，同時可以減低聚焦點外不必要的光破壞，並且加強了掃描影像的深度範圍，因此非常適用於三維活體組織的影像掃描。其中，膠原蛋白在脊椎動物組織內，是含量最豐富的蛋白質之一。因此發展其適當的量測工具並使其可應用於生物物理、生理學、以及生醫工程領域是非常重要的。在此項研究中，我們結合了多光子、二倍頻、以及二階電化率等非線性技術去量測並觀察膠原蛋白在各組織內的結構及成分，並動態的觀察膠原蛋白在軟骨組織工程中的增生情形。
首先利用正向與反向二倍頻訊號觀察眼角膜在正常與水腫情況下，其膠原蛋白結構在大範圍且不同深度的相對變化。實驗觀察後發現，角膜水腫時其膠原蛋白薄層間隙會不規則的增加，同時薄層厚度在後段(深度>200 μm)也會明確的增加，而前段的薄層可能因為其結構上包含較多交錯的膠原蛋白纖維在厚度上並沒有明顯的改變。而此項研究也顯示出二倍頻顯微術可以有效的提供組織內膠原蛋白的結構資訊同時未來也可望被應用在活體上研究角膜水腫的相關議題。
由於電化率可以進一步用來判斷膠原蛋白螺旋角，因此二階電化率顯微術在研究中也被應用作為分辨不同種類膠原蛋白的機制之一。以第一型膠原蛋白為例，我們從大鼠尾腱中量測到第一型膠原蛋白的二階電化率比值為Xzzz/Xzxx=1.41±0.08 ，Xzzz/Xzxx=0.77±0.11；從大鼠氣管軟骨組織量測到的第二型膠原蛋白為Xzzz/Xzxx=1.16±0.13，Xzzz/Xzxx=10.40±0.14；在大鼠皮膚中的第三型膠原蛋白所量測到的二階電化率為Xzzz/Xzxx=1.20±0.14，Xzzz/Xzxx=0.49±0.15。為了更進一步確認所量測到的電化率張量非受到相關激發波長所影響，我們量測了在不同激發波長下第一型與第二型膠原蛋白的電化率比值也證明在此範圍下膠原蛋白的量測是不受共振之影響的。
由於在多數生物組織中，不同型膠原蛋白常共同存在。我們利用高斯混和模型擬合二階電化率在組織中的分佈藉以量測不同型態膠原蛋白在組織中的相對比例。在皮膚組織中，利用二階電化率顯微術量測第一型與第三型膠原蛋白的比例與傳統生化方法量測之結果是非常吻合的。此項方法結合多光子顯微術也成功的觀測到骨隨間葉幹細胞分化成軟骨組織過程中，第二型與第一型膠原蛋白之相對比例隨著時間的變化。因此，多光子、二倍頻、二階電化率等技術之結合顯示其未來應用在偵測生物組織的特性並且即時的觀察組織工程中膠原蛋白的結構的高度發展性。

關鍵字

二倍頻 ； 膠原蛋白 ； 二階極化率 ； 多光子顯微術

並列摘要

Nonlinear optical microscopy has been the preferred technique in a wide array of bioimaging applications. Intrinsic optical sectioning, out-of-focus photo damage reduction, and enhanced image depths facilitated three-dimensional imaging of tissues in vivo. Since collagen is the most abundant proteins in vertebrates, developing an imaging technique to study the structure and function of collagen-containing tissues is of significant values in biophysics, physiology, and biomedical engineering. In this thesis, multiphoton, second harmonic generation (SHG), and second order susceptibility (SOS) microscopy were used as to image and study dynamics of collagen structure in different tissues, including collagen production in chondrogenic tissue engineering.
We first utilized SHG microscopy to investigate the structural features of corneal edema by simultaneously collecting forward and backward SHG signals from normal and over-hydrated bovine cornea over a large area and at different depths. For edematous cornea, the uneven expansion in lamellar interspacing and also, increased lamellar thickness in posterior stroma (depth > 200 μm) were identified, while the anterior stroma composed of interwoven collagen architecture remained unaffected. This work demonstrates the capability of SHG imaging in providing morphological information for the investigation of corneal edema biophysics and that this approach may be applied in the evaluation of advancing corneal edema in vivo.
Secondly, second order susceptibility (SOS) microscopy was used as a contrast mechanism for distinguishing collagen microstructure. For type I collagen, the SOS ratios, χzzz/χzxx =1.41±0.08 and χxzx/χzxx =0.77±0.11, were obtained from rat tail tendon. χzzz/χzxx =1.16±0.13 and χxzx/χzxx =0.40±0.14 were obtained for type II collagen from rat trachea cartilage. χzzz/χzxx =1.20±0.14 andχxzx/χzxx =0.49±0.15 were obtained for type III collagen from rat skin. Sinceχzzz/χzxx is related to the pitch angle of collagen triple helix, these results imply that the variant or similar chi tensor ratio between different collagen types was mainly dependent on the hetero- or homotrimer structures. Furthermore, experiment to measure the dependence of the SOS on different excitation wavelength (725-875 nm) was performed in collagen I and II, which shows constant SOS ratio and exhibits the off-resonance state of collagen within the wavelength region.
Considering different collagen type mixture appeared in most tissues, we introduced a method to determine the relative proportion of collagen type by analyzing the histogram of SOS ratio. The highly coincidence of collagen III/I ratio through SOS and biochemistry analysis was demonstrated in rat skin tissue. Combined multiphoton microscopy (MPM), the qualitative progress of chondrogenic differentiation of human mesenchymal stem cells was followed. The applications of SOS and MPM in biological specimens show the potential of this methodology in detecting the quality of tissues and the structural dynamics of engineered tissues.

並列關鍵字

second harmonic ； collagen:second order susceptibility ； multiphoton microscopy

參考文獻

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