School of Physical Sciences, Department of Physics, Multidisciplinary Nanotechnology Centre, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Ultramicroscopy. 2011 Jul;111(8):1200-5. doi: 10.1016/j.ultramic.2011.01.008. Epub 2011 Feb 1.
Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy from cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The application of the technique to investigate cell-cell adhesion has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions. This has been achieved via the selective tagging of the cell adhesion protein, E-cadherin, by immunofluorescence labelling. Two labelling routes were explored; Alexa Fluor 488 and semiconductor quantum dots. The quantum dots demonstrated significantly enhanced photostability and high quantum yield making them a versatile alternative to the conventional organic fluorophores often used in such a study. Analysis of individual cells revealed that E-cadherin is predominantly located along the cell periphery but is also found to extend throughout their filopodia. We have demonstrated that with a fully optimised sample preparation methodology, quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.
扫描近场光学显微镜 (SNOM) 已被用于从细胞膜上同时获取高分辨率荧光图像和剪切力原子力显微镜图像。实现这种技术克服了标准荧光显微镜中存在的光学衍射限制，同时还提供了重要的形貌信息。该技术在研究细胞-细胞黏附中的应用揭示了丝状伪足的相互作用及其在建立黏着连接中的功能关系。这是通过免疫荧光标记对细胞黏附蛋白 E-钙黏蛋白进行选择性标记来实现的。探索了两种标记途径；Alexa Fluor 488 和半导体量子点。量子点表现出显著增强的光稳定性和高光量子产率，使它们成为此类研究中常用的传统有机荧光团的多功能替代品。对单个细胞的分析表明，E-钙黏蛋白主要位于细胞周围，但也发现它延伸到整个丝状伪足中。我们已经证明，通过完全优化的样品制备方法，结合 SNOM 成像的量子点标记可以成功地应用于研究精细细胞膜内生物分子的定位。
