生物系统中金纳米粒子的高灵敏度光学探测与成像

Nanometer sized particles can easily go through the membrane of cells due to the size advantage, which make the explorations of biology and life mechanism possible even into cell level by tracing and imaging a single nanoparticle. Fluorescence based optical methods play a dominant role in those studies. However the photobleaching or "blinking" associate respectively with organic fluorescent particles or quantum dots seriously limited the observation time. Gold nanopartilces provide superior alternatives to fluorescent markers with unique and highly desirable optical properties such as high brightness, biocompability, no photobleaching, no blinking, and an unlimited observation time. The tunability of the optical response relies on the fact that optical properties of individual particle are dependent strongly on their engineered nanostructures. The development of optical techniques that can detect and characterize the optical response of a single gold nanostructure is of central interest in both fundamental and applied nanoscience. The ability to probe within thick samples makes the far-field optical approach more attracitve in potential cells applications. However it holds a great challenge to optically detect a nanometer sized gold particle due to the very weak signal. The detection in a biological environmental makes the work even harder due to the strong scattering background interference from the dielectric particles. Holding the aim to detect nanometer sized gold particles in biological environment, we will carry out the following studies. In this project we propose a novel optical far field detection approach which can realize the ultrasensitive detection of a single gold nanoparticle. With the assistant of this detection approach, we will explore the unique optical properties of each individual gold nanostructures including spherical nanoparticles, nanrods and core-shell nanoparticles by their illustrations in the amplitude and phase microscopy images. Especially the conditions led to localized surface Plasmon resonance (LSPR) associate with each nanostructures will be stressed by numberical simulation and experimental measurement. Based on the enhancement of LSPR to the amplitude signal, a creative method is proposed to distinguish gold nanoparticles from other dielectric particles normally existed in the biological environment. To further explore the optimum nanostructures which are applicable to meet different biological environment requirements, polystyrene micro- or nano-spheres are chosen to mimic the absorption and scattering properties of different biological environments.

纳米粒子由于尺寸上的优势可以直接穿透细胞壁，因而通过对单个粒子的跟踪和成像可使对生物/生命机理的科学探索发展到细胞层面上，是当今国际前沿课题。金纳米粒子展现的独特光学性质使得光学探测手段备受关注。发展光学远场技术对于深入到厚样品（如细胞）中探测金纳米粒子具有重要研究价值。然而用光学方法探测纳米级的金粒子是个极大的挑战，因为信号极其微弱。本申请在前期工作的基础上研究超高灵敏度的金纳米粒子的光学远场探测和显微成像方法；在"光学可见"的支持下，研究金纳米粒子的光学性质随微观结构参数的变化规律在显微图像上的表征和增强；在此基础上利用其独特的增强特性研究在有其他散射粒子干扰的环境中提取和识别金粒子的方法，并以聚苯乙烯球模拟不同吸收波段和散射条件的生物环境，探索金纳米粒子在不同生物环境中的最佳结构参数。本申请旨在于通过对金纳米粒子光学性质的研究来探索在生物环境中的单个金粒子的光学探测、识别和定位方法。

金纳米粒子以其高亮度、无漂白、良好的生物兼容性、观测寿命长等优点是荧光物质的理想替代物。但是由于纳米物体的散射面极小，使得从强大的入射背景中提取其散射信号进行探测变得极为困难。本项目在前期工作的基础上1）建立了基于激光扫描共焦原理的正交偏振外差光学显微成像系统实现了单个金纳米粒子的高灵敏度光学成像，并在此灵敏度的支持下将探测目标延伸到普通介质材料，实现了介质微纳米粒子的无标记直接光学成像。普通介质微纳粒子具有与生物粒子相似的光学性质，例如粒子的直径在100nm-400nm是病毒的典型尺寸范围，该方法的建立可在无需标记的情况下，直接对该类粒子进行探测，从而可早期发现、早期控制传染源，在公共健康防护方面具有很好的前景。2）基于时域有限差分法和Mie散射理论建立了高会聚光场下光与微纳物质作用的散射远场显微成像仿真模型，该模型与实验结果高度吻合。粒子与光相互作用后，其散射光的波前携带粒子本身形貌的特征，这些特征在本项目所建立的系统中通过散射光的振幅和相位图像的形式表现出来，该理论模型能够对该系统所测量得到的独特的影像信息进行充分解读，理解其底层的物理机理，并确定了通过振幅和相位图像获得粒子尺寸的方法。3）基于时域有限差分法建立了金纳米粒子的光谱光学特性的计算模型，研究了其峰值局部表面等离子体共振波长（LSPR）随结构参数的变化规律。通过改变粒子的尺寸、形状、间距等微观结构参数，分别对实心、芯壳形、芯帽形等不同结构的单双粒子进行了仿真计算。发现芯帽形双粒子所产生的LSPR波长红移和消光增强甚至高于芯壳形粒子对，目前该类粒子的研究还先有报道，本课题组已开展后续的制备等研究。4）基于上述的研究，利用金纳米粒子在其LSPR 峰值波长处所产生的独特消光增强，建立了双波长激发剔除大分子干扰的金粒子识别与提出的方法。

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