Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
Nanoscale. 2011 Sep 1;3(9):3567-72. doi: 10.1039/c1nr10182j. Epub 2011 Jun 1.
Mapping of individual ligand molecules and their binding sites in single protein-ligand complexes at nanometer resolution in real-time would enable probing their structures and functions in vitro and in vivo. In this study, we have developed far-field photostable optical nanoscopy (PHOTON) for mapping single ligand molecules (biotin) and their binding sites in individual protein-ligand complexes (streptavidin-biotin) with 1.2 nm spatial resolution and 100 ms temporal resolution. PHOTON includes one standard far-field optical microscope with a halogen-lamp illuminator; single-molecule-nanoparticle-optical-biosensors (SMNOBS) with exceptionally high quantum-yield (QY) of Rayleigh scattering and photostability (non-photobleaching, non-photoblinking) as imaging probes; and Multispectral Imaging System (MSIS) for spectral isolation of individual SMNOBS with 1 nm wavelength resolution. Intrinsic size- and shape- dependent localized-surface-plasmon-resonance (LSPR) spectra of single SMNOBS provide multiple-spectral (color) nanoprobes for sub-diffraction imaging, offering feasibility of probing of binding structures and functions of single protein-ligand complexes at nm (potentially achieving Ångstrom) resolution in real-time.
在纳米分辨率实时水平上，对单个蛋白-配体复合物中单个配体分子及其结合位点进行测绘，将能够在体外和体内探测它们的结构和功能。在本研究中，我们开发了远场光稳定光学纳米显微镜（PHOTON），用于测绘单个配体分子（生物素）及其在单个蛋白-配体复合物（链霉亲和素-生物素）中的结合位点，空间分辨率为 1.2nm，时间分辨率为 100ms。PHOTON 包括一个带有卤素灯照明器的标准远场光学显微镜；单分子纳米粒子光学生物传感器（SMNOBS），其瑞利散射的量子产率（QY）和光稳定性（非光漂白、非光闪烁）极高，用作成像探针；以及多光谱成像系统（MSIS），用于对具有 1nm 波长分辨率的单个 SMNOBS 进行光谱分离。单个 SMNOBS 的固有尺寸和形状依赖性局域表面等离子体共振（LSPR）光谱提供了多个光谱（颜色）纳米探针，用于亚衍射成像，为实时在 nm（可能达到 Ångstrom）分辨率下探测单个蛋白-配体复合物的结合结构和功能提供了可行性。
