以偏振二倍頻顯微術分析膠原蛋白結構

Abstract

在光學顯微術中，使用線性偏振光掃描生物的纖維樣品，可以得到其分子尺度上的資訊，像是肌肉、膠原蛋白束等。但是在雷射掃描顯微鏡中，光的偏振性常會被光學元件所改變，如分光鏡；於是我們使用半波長片和四分之一波長片來校正這種偏振性的改變，並在反射式雷射顯微鏡中以改變線偏振光的角度，取得老鼠尾巴的二倍頻影像。從二倍頻強度的變化，可以得到老鼠尾巴內膠原蛋白的二階極化張量，並且以我們旋轉入射光的方法，可以減少轉動樣品所帶來的困擾。藉由螺旋結構來模擬膠原蛋白分子，偏振光的二倍頻影像可以得到分子尺度上的訊息；從強度對於入射光偏振性的改變上，我們得到的極化張量可以判斷出膠原蛋白的螺旋角，而且與原子力顯微鏡的結果相符。對於非中心對稱的生物樣品，我們證明了偏振化的二倍頻顯微術可以在型態跟強度之外，提供額外分子上的資訊，並且具有生醫應用上的潛力。

In imaging anisotropic samples with optical microscopy, the use of controlled, polarized light source can be used to gain molecular information of fibrous materials such as muscle and collagen fibers. However, the delivery of polarized excitation light source in a system such as a laser scanning optical microscope often encounter the problem of the polarization ellipticity altering effect of the main dichroic mirror and other optical components. By using a half-wave plate and a quarter-wave plate, we demonstrated that the polarization ellpticity altering effect of the dichroic mirror in an epi-illuminated laser scanning microscope can be corrected for, and that this approach may be used to obtain polarized second harmonic generation (SHG) images of rat tail tendon using controlled polarized excitation. In this work, the excitation polarization angular dependence of the SHG intensities were fitted to a model to determine the ratio of the second order susceptibility tensor elements associated with type I collagen in rat tail tendon and our methodology can be applied to polarized SHG imaging without the disadvantages of the sample rotation approach. With the helix structure assumption, we obtained collagen molecular information by polarized SHG images. By analyzing the susceptibility tensor using the measured intensity variation with polarization of incident light, we determined the pitch angle of collagen. Our result is consistent with that obtained using atomic force microscope. We showed that polarized SHG has a great potential in imaging noncentrosymmetric biological sample, giving information on the molecular scale in addition to morphological informations.