Institute for Integrated Cell-Material Sciences, 2 Institute for Frontier Medical Sciences, and 3 Department of Materials Science and Engineering, Kyoto University, Kyoto 606-8501, Japan.
J Cell Biol. 2013 Sep 16;202(6):967-83. doi: 10.1083/jcb.201301053.
Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed.
荧光显微镜在细胞生物学和生物医学研究中得到了广泛的应用，但目前可用的荧光探针存在着三个主要问题：光漂白、闪烁和尺寸较大。我们通过开发具有 4.1nm 水动力直径的生物相容性、红色发射硅纳米晶体（SiNCs），特别关注单分子成像，解决了这些问题。我们开发了通过简单的化学蚀刻生产 SiNCs、亲水性涂层以及精确地将它们以 1:1 比例偶联到生物分子的方法。在视频速率下观察到的 300 分钟总时间内，单个 SiNCs 既没有闪烁也没有光漂白。活细胞的质膜中的单个受体分子（使用转铁蛋白受体）被成像的时间超过了其他探针的 10 倍以上，这使得首次有可能在单分子水平上观察到受体分子的内化过程。揭示了在 1-2 µm 尺度上的分子扩散率的空间变化，即在质膜中出现更高水平的域镶嵌现象。
