Department of Micro Engineering, Kyoto University, Kyoto, Japan.
Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Aichi, Japan.
Methods Mol Biol. 2022;2430:121-131. doi: 10.1007/978-1-0716-1983-4_8.
Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM. This concentration is much lower than the concentration of adenosine triphosphate (ATP) in vivo, hindering the single-molecule observation of fluorescently labeled ATP hydrolyzed by motor proteins under the physiologically relevant conditions. Here, we provide a method for the use of single-molecule fluorescence microscopy in the presence of ~500 nM of fluorescently labeled ATP. To achieve this, a device equipped with nano-slits is used to confine excitation light into its slits as an expansion of zero-mode waveguides (ZMWs). Conventional ZMWs equip apertures with a diameter smaller than the wavelength of light to suppress background noise from the labeled molecules diffusing outside of the apertures. While they are not compatible with filamentous objects, our linear-ZMWs enable the usage of filamentous objects, such as microtubules. An experiment using linear-ZMWs demonstrated the successful exploration of the interaction between kinesin and ATP using single-molecule fluorescence microscopy.
单分子荧光显微镜是研究微管相关马达蛋白（如驱动蛋白）的化学机械偶联的关键工具。然而，实施单分子观察的一个主要限制因素是荧光标记分子的浓度。例如，在全内反射荧光显微镜中，可用的浓度约为 10 nM。这个浓度远低于体内三磷酸腺苷 (ATP) 的浓度，这阻碍了在生理相关条件下对荧光标记的 ATP 水解的马达蛋白进行单分子观察。在这里，我们提供了一种在约 500 nM 的荧光标记 ATP 存在下使用单分子荧光显微镜的方法。为了实现这一点，使用带有纳米狭缝的设备将激发光限制在狭缝中，作为零模波导 (ZMW) 的扩展。传统的 ZMW 配备的孔径小于光的波长，以抑制标记分子从孔径外扩散产生的背景噪声。虽然它们与丝状物体不兼容，但我们的线性 ZMW 可以使用丝状物体，如微管。使用线性 ZMW 的实验证明了使用单分子荧光显微镜成功探索了驱动蛋白和 ATP 之间的相互作用。
