Contribution of Chirality and Anisotropy for Nonlinear Optical Activity in Collagenous Tissues

• Main Authors: Mei-Yu Chen, 陳美瑜
• Other Authors: 朱士維
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/79120713195794034565

博士 === 國立臺灣大學 === 物理研究所 === 103 === Nature prefers chiral molecules so the chirality studies have always been a very hot topic. Natural chiral compounds such as DNA, proteins, peptides, carbohydrates, etc. require correct handedness in chemical recognition and interaction. Conventionally, to study chirality, circular dichroism (CD) is the most popular technique. However, CD spectroscopy is limited by its poor signal contrast and relatively inadequate chiral structure information, so previous studies are constrained to surfaces or purified bulk materials. It is highly desirable to study chirality in complex biological molecules under native microenvironment. Therefore, we aim to develop techniques that can overcome the limitations of the linear CD method. In this dissertation, we study two nonlinear optical activity approaches to address these limitations. The first technique is second harmonic generation circular-dichroism (SHG-CD), which detects variation of second harmonic generation signals for left- and right-circularly polarized light. The main advantages of SHG-CD is that the signal contrast reaches unity. The second one is circular intensity difference (CID) derived from Raman optical activity (ROA), which measures a small difference in the intensity of vibrational Raman scattering from chiral molecules with right- and left-circularly polarized light. The outstanding property of ROA is that the molecular bond chirality can be unraveled via vibrational modes analysis. In our study of SHG-CD, we achieve several accomplishments which will be vital in the future to study of chirality in complex tissue samples. First, we found that SHG-CD response sensitively depends on two main factors, intrinsic chirality and molecular anisotropy, the latter of which is not possible to avoid when studying chiral molecules under native microenvironment. Furthermore, we verify that chirality-induced SHG-CD is significantly enhanced at molecular resonance, while anisotropy-induced SHG-CD kept roughly the same at every wavelength. Second, SHG-CD shows much better chiral contrast than traditional chiroptical techniques in thick tissue. Third, SHG-CD provides good optical sectioning capability that is suitable for three dimensional imaging application. To further explore the molecular origins of chirality, we use CID spectroscopy to investigate chiral sensitive chemical bonds of the entire collagen molecular structure. Our main achievements are the following. First, to our knowledge we are the pioneer to report CID spectra of type I collagen within a native tendon tissue. Second, we find the largest CID signals occur at the wavenumbers of 938 cm−1 (α helix), 1246-1271 cm−1 (Amide III), and 1668cm−1 (Amide I), which are all associated with vibrational modes related to the collagen backbone. In contrast, 2944 cm−1 (amino acids side chains) exhibits very large Raman signals, but diminishing CID response. Therefore, our study verify that the chiral response of collagen is dominated by the stereo organization of the backbone. That is, CID spectra extracts the information about the main chiral sources which cannot be provided by using any other conventional measurement. Third, our CID values are one order of magnitude larger than theoretical estimation from isotropically distributed molecules. The reason is that type-I collagen molecules arrange orderly in the tissue, resulting in significant anisotropy, which in turn leads to coherent summation of Raman signals. Fourth, we find that the sign of CID values from a single collagen fiber alters from positive to negative, manifesting the influence of anisotropy and fiber orientation. Fifth, we demonstrate CID microscopic images, which can provide not only direct visualization of molecular chirality, but also molecular specificity from Raman signature in intact biological tissues. We are the first group in the world to use SHG-CD and CID microspectroscopyto identify intrinsic chirality and chiral sensitive chemical bondsof the type I collagen tissue samples in situ. Our investigation constitutes an important landmark towards a realistic chiralspectroscopic/microscopic technique and allowed us to expand our current knowledge of the standard spectral characteristic of the classical, long-range, three-dimensional structures of polypeptides and proteins in complex structures noninvasively.