Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)
Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)
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DOI：
10.1364/oe.21.001189
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发表时间：
2013-01-14
期刊：
影响因子：
3.8
通讯作者：
Spudich, James A.
中科院分区：
文献类型：
--
作者：
Elting, Mary Williard;Leslie, Sabrina R.;Spudich, James A.
在存在高荧光团浓度的情况下解析单个荧光分子仍然是单分子生物物理学中的一个挑战，它限制了我们对弱分子相互作用的理解。全内反射荧光（TIRF）成像是单分子荧光显微镜的主力，可以在高达约100 nM的浓度下进行实验，但许多生物相互作用具有相当弱的亲和力，因此需要至少一个物种处于微摩尔或更高的浓度。目前TIRF的替代方案通常需要三维约束，因此对于扩展底物，如细胞骨架细丝，可能存在问题。为了解决这一挑战，我们展示并应用了两种新的单分子荧光显微镜技术，线性零模波导（zmw）和凸透镜诱导约束（CLIC），用于成像分子马达肌凝蛋白V和VI沿着肌动蛋白细丝的过程运动。这两种技术都可以在比TIRF显微镜更高的荧光团浓度下进行成像。它们将使沿着丝状轨迹运动的一系列进程分子马达（如其他肌凝蛋白、动力蛋白和运动蛋白）的生物物理测量成为可能。这些技术的一个特别突出的应用将是通过直接成像荧光核苷酸分子在通过肌动蛋白或微管轨迹时与进程马达相互作用来检查化学-力学耦合。(C) 2013年美国光学学会
Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks. (C) 2013 Optical Society of America