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首页> 外文学位 >Spectral fluorescence measurements on reflecting surfaces shedding light onto conformation and orientation of macromolecules.
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Spectral fluorescence measurements on reflecting surfaces shedding light onto conformation and orientation of macromolecules.

机译：反射表面上的光谱荧光测量，使光散射到大分子的构象和方向上。

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The functionality of macromolecules depends strongly on their structural arrangements that form the functional sites and on the accessibility of these sites. These molecules are often immobilized on surfaces to study their interactions with other molecules, in biological and medical sciences. When macromolecules are in contact with a surface, a new structural arrangement, or conformation, may be adopted due to free energy minimization mechanisms, and the functional sites may be rendered inactive. Additionally, the molecular orientation acquired for surface attachment may hinder the accessibility of the functional sites. Therefore, conformation and orientation of macromolecules are of utmost importance to sustain their functional behavior on a surface and require systematic studies. However, the length scale necessary to directly measure macromolecular conformation is far beyond the capability of conventional optical microscopy.;We have developed a spectroscopy technique to study the surface conformation and orientation of fluorescently tagged macromolecules. The technique, spectral self-interference fluorescence microscopy (SSFM), utilizes the spectral oscillations in the radiated intensity due to the interference of direct and reflected fluorescence from emitters near a dielectric surface. Using the sub-nanometer emitter axial localization capability of this technique, we have determined the conformational change of surface adsorbed polymer chains upon hydration. The results revealed the importance of this conformational change in the efficiency of the polymeric structure for surface binding of probes in microarray applications to study ligand-analyte interactions.;SSFM in the traditional configuration lacked high lateral resolution due to the low numerical aperture (NA) requirement to achieve sub-nanometer axial localization. However, for applications that involve resolving finer structures such as topographical mapping of cell surfaces or study of sub-cellular components, high lateral resolution is required. Using high-NA objectives results in a diminished fringe contrast of spectral oscillations, and thus the localization capability is reduced. Through angular tailoring of the radiated intensity, we have achieved sub-micron lateral resolution while maintaining sub-nanometer axial localization capability. The elimination of the low-NA requirement with this angular tailoring approach also opened up the possibility of determining the dipole orientation information from the spectral signature in addition to the axial position information. Simulations demonstrate that the dipole orientation of the emitters can be detected with a high precision.
机译：大分子的功能在很大程度上取决于其形成功能位点的结构安排以及这些位点的可及性。在生物学和医学科学中，这些分子通常固定在表面上，以研究它们与其他分子的相互作用。当大分子与表面接触时，由于自由能最小化机理，可以采用新的结构排列或构象，并使功能位点失活。另外，为表面附着而获得的分子取向可能会阻碍功能位点的可及性。因此，大分子的构象和取向对于维持其在表面上的功能行为至关重要，需要进行系统的研究。然而，直接测量大分子构象所必需的长度尺度远远超出了常规光学显微镜的能力。我们已经开发了一种光谱技术来研究荧光标记的大分子的表面构象和取向。光谱自干扰荧光显微镜（SSFM）技术利用了辐射强度中的光谱振荡，这是由于电介质表面附近的发射器产生的直接和反射荧光的干扰所致。使用该技术的亚纳米发射器轴向定位能力，我们确定了水合后表面吸附的聚合物链的构象变化。结果揭示了这种构象变化对于微阵列应用中探针表面结合研究配体-分析物相互作用的聚合物结构效率的构象变化的重要性。由于低数值孔径（NA），传统配置中的SSFM缺乏高横向分辨率要求实现亚纳米级的轴向定位。但是，对于涉及解析更精细结构的应用（例如细胞表面的地形图绘制或亚细胞成分的研究），需要高横向分辨率。使用高NA物镜会降低光谱振荡的条纹对比度，从而降低定位能力。通过对辐射强度进行角度调整，我们在保持亚纳米轴向定位能力的同时，实现了亚微米级的横向分辨率。用这种角度调整方法消除了低NA的要求，除了轴向位置信息之外，还开辟了从频谱特征确定偶极子定向信息的可能性。仿真表明，可以高精度检测发射器的偶极子方向。

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