利用偏振二倍頻顯微術分析第一類與第二類膠原蛋白的結構

Structural Analysis of Type I and Type II Collagen by Polarized Second Harmonic Generation Microscopy

李宗憲(Tsung-Hsien Li)

指導教授 ： 董成淵

國立臺灣大學/理學院/物理研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01145

摘要

膠原蛋白是人體中非常重要的一種蛋白質，其分子由三條多肽鏈纏繞而成，具有三股螺旋結構。依據多肽鏈中的胺基酸序列的不同，可以將目前已知的膠原蛋白分為29種類型。
光學顯微術中，利用偏振二倍頻顯微術來掃描生物樣品，可以得到其分子尺度上的資訊，但考慮到光學元件，如分光鏡，會改變光的偏振性。因此，我們使用了二分之一波長片及四分之一波長片來彌補這種效應，同時也藉此旋轉雷射的偏振角度。如此一來，我們校正了分光鏡造成的偏振性改變，也減少了轉動樣品所帶來的困擾。
藉由分析隨著入射光的偏振角度而改變的二倍頻強度變化，我們可以分析出生物樣品內膠原蛋白的二階極化率張量，並且推導出膠原蛋白分子的螺旋角。此實驗中，我們使用了大鼠尾巴的肌腱和豬腿骨間的關節軟骨來做比較，他們分別含有大量的第一類及第二類的膠原蛋白，而這兩種不同類型的膠原蛋白分析出來的二階極化率張量及螺旋角確實有所不同！第一類和第二類膠原蛋白分子的螺旋角分別為49.1°±1.3° and 52.8°±2.7°。證明了偏振二倍頻顯微術能夠有效的分辨第一類及第二類的膠原蛋白。

關鍵字

二倍頻 ； 膠原蛋白 ； 偏振 ； 肌腱 ； 軟骨 ； 非線性極化率 ； 螺旋結構 ； 螺旋角

並列摘要

Collagen is one of the most important and abundant protein in human body. Its molecules composed of three entwined polypeptide chains. There are currently 29 known types of collagen differentiated by the sequence of residues in each polypeptide chain.
Utilizing polarized second harmonic generation microscopy, we can obtain structural information at the molecular level. In order to compensate the depolarization effects of optical component such as the dichroic mirror, we used the combination of a half-wave plate and a quarter-wave plate to rotate the polarization angle of the laser incident on the sample. This allows us to perform polarization microscopy without the drawbacks of sample rotation.
By measuring second harmonic generation intensity variation as a function of the polarization angle of the incident light, we can analyze the second-order susceptibility tensor of collagen in a biological sample and determinate the pitch angle of the collagen helix. In this study, we compared the second-order susceptibility tensor of collagen from rat tail tendon and cartilage of porcine femoral-tibial joint, which contain abundant type I and type II collagens respectively. We found that the second-order susceptibility tensor and the calculated pitch angle for type I and II collagen to be 49.20°±0.34° and 52.43°±0.72°. Our results suggest that polarized SHG microscopy could potentially be used for distinguishing these two types of collagen.

並列關鍵字

second harmonic generation ； collagen ； polarization ； tendon ； cartilage ； nonlinear susceptibility ； helical structure ； pitch angle

參考文獻

1. Plotnikov, S.V., Millard, A.C., Campagnola, P.J. & Mohler, W.A. Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres. Biophysical Journal 90, 693-703 (2006).

2. Nadiarnykh, O., LaComb, R., Campagnola, P.J. & Mohler, W.A. Coherent and incoherent SHG in fibrillar cellulose matrices. Optics Express 15, 3348-3360 (2007).

3. Zoumi, A., Yeh, A. & Tromberg, B.J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proceedings of the National Academy of Sciences of the United States of America 99, 11014-11019 (2002).

4. Brown, E.B., et al. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nature Medicine 7, 864-868 (2001).

5. Liu, Y., et al. Visualization of hepatobiliary excretory function by intravital multiphoton microscopy. Journal of Biomedical Optics 12, 014014 (2007).