Zhao Yanhui, Chen Danqi, Yue Hongjun, Spiering Michelle M, Zhao Chenglong, Benkovic Stephen J, Huang Tony Jun
Department of Engineering Science and Mechanics and ‡Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Nano Lett. 2014;14(4):1952-60. doi: 10.1021/nl404802f. Epub 2014 Mar 24.
The ability of zero-mode waveguides (ZMWs) to guide light energy into subwavelength-diameter cylindrical nanoapertures has been exploited for single-molecule fluorescence studies of biomolecules at micromolar concentrations, the typical dissociation constants for biomolecular interactions. Although epi-fluorescence microscopy is now adopted for ZMW-based imaging as an alternative to the commercialized ZMW imaging platform, its suitability and performance awaits rigorous examination. Here, we present conical lens-based dark-field fluorescence microscopy in combination with a ZMW/microfluidic chip for single-molecule fluorescence imaging. We demonstrate that compared to epi-illumination, the dark-field configuration displayed diminished background and noise and enhanced signal-to-noise ratios. This signal-to-noise ratio for imaging using the dark-field setup remains essentially unperturbed by the presence of background fluorescent molecules at micromolar concentration. Our design allowed single-molecule FRET studies that revealed weak DNA-protein and protein-protein interactions found with T4 replisomal proteins.
零模波导（ZMWs）将光能引导至亚波长直径圆柱形纳米孔的能力已被用于对微摩尔浓度生物分子进行单分子荧光研究，这是生物分子相互作用的典型解离常数。尽管落射荧光显微镜目前已被用作基于ZMW成像的商业化ZMW成像平台的替代方法用于基于ZMW的成像，但其适用性和性能仍有待严格检验。在此，我们展示了基于锥形透镜的暗场荧光显微镜与ZMW/微流控芯片相结合用于单分子荧光成像。我们证明，与落射照明相比，暗场配置显示出背景和噪声减少，信噪比提高。使用暗场设置成像的信噪比基本上不受微摩尔浓度背景荧光分子存在的影响。我们的设计允许进行单分子FRET研究，揭示了T4复制体蛋白中存在的弱DNA-蛋白质和蛋白质-蛋白质相互作用。
