Sowa Yoshiyuki, Steel Bradley C, Berry Richard M
Department of Frontier Bioscience, Hosei University, Koganei, Tokyo 184-8584, Japan.
Rev Sci Instrum. 2010 Nov;81(11):113704. doi: 10.1063/1.3495960.
Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ~0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells.
光学显微镜下观察单分子技术的最新进展有助于揭示分子机器的工作机制。根据研究兴趣，可使用多种标记物来检测分子，从单个荧光团到微米大小的标记物。在此，我们展示了一种新型且简单的物镜型背散射显微镜，可用于以纳米和微秒分辨率追踪金纳米颗粒。我们系统在55千赫兹带宽下的总噪声约为每轴0.6纳米，足以测量分子运动。我们发现我们的背散射显微镜不仅对体外实验有用，对体内实验也有用，因为与传统暗场显微镜相比，细胞的背景散射更低。我们展示了该技术在测量活的大肠杆菌细胞中生物旋转分子马达——细菌鞭毛马达运动方面的应用。
