Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking
Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking
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DOI：
10.1529/biophysj.107.104737
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发表时间：
2007-11-01
影响因子：
3.4
通讯作者：
Klenerman, David
中科院分区：
文献类型：
--
作者：
Bruckbauer, Andreas;James, Peter;Klenerman, David
我们已经开发了一种新的方法，使用纳米移液管，用于控制电压驱动的个人荧光标记的探针分子传递到质膜，我们用于单分子荧光跟踪（SMT）。该方法的优点是：1）将探针应用于膜上的预定区域; 2）仅将一个或几个分子释放到细胞表面上; 3）当与全内反射荧光显微镜结合时，由于未结合的分子，背景非常低;以及4）首先优化实验，然后在同一细胞上重复实验的能力。我们验证了该方法进行SMT研究的扩散与Atto 647-麦胚凝集素标记的单个膜糖蛋白在不同的表面结构域的猪精子。我们发现与布朗扩散的偏差很小，顶体区的平均扩散系数为0.79 +/- 0.04 μ m（2）/s，顶体后区的平均扩散系数为0.10 +/- 0.02 μ m（2）/s;这种差异可能反映了不同的膜结构。我们还表明，我们可以分析细胞膜的不同子区域的扩散特性，并探测扩散屏障的存在。将这种新方法扩展到其他探针和细胞应该是简单的，并且它可以用作研究细胞膜的新工具。
We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking ( SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 +/- 0.04 mu m(2)/s in the acrosomal region and 0.10 +/- 0.02 mu m(2)/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.