用于全内反射荧光显微镜的盖玻片清洗与功能化处理
Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy.
作者信息
Kudalkar Emily M, Deng Yi, Davis Trisha N, Asbury Charles L
机构信息
Department of Biochemistry, University of Washington, Seattle, Washington 98195;
Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195.
出版信息
Cold Spring Harb Protoc. 2016 May 2;2016(5):pdb.prot085548. doi: 10.1101/pdb.prot085548.
Total internal reflection fluorescence (TIRF) microscopy allows visualization of biological events at the single-molecule level by restricting excitation to a precise focal plane near the coverslip and eliminating out-of-focus fluorescence. The quality of TIRF imaging relies on a high signal-to-noise ratio and therefore it is imperative to prevent adherence of molecules to the glass coverslip. Nonspecific interactions can make it difficult to distinguish true binding events and may also interfere with accurate quantification of background noise. In addition, nonspecific binding of the fluorescently tagged protein will lower the effective working concentration, thereby altering values used to calculate affinity constants. To prevent spurious interactions, we thoroughly clean the surface of the coverslip and then functionalize the glass either by applying a layer of silane or by coating with a lipid bilayer.
全内反射荧光（TIRF）显微镜通过将激发限制在盖玻片附近的精确焦平面并消除离焦荧光，从而能够在单分子水平上可视化生物事件。TIRF成像的质量依赖于高信噪比，因此必须防止分子粘附到玻璃盖玻片上。非特异性相互作用可能难以区分真正的结合事件，也可能干扰背景噪声的准确量化。此外，荧光标记蛋白的非特异性结合会降低有效工作浓度，从而改变用于计算亲和常数的值。为了防止虚假相互作用，我们彻底清洁盖玻片表面，然后通过涂覆一层硅烷或用脂质双层包被来对玻璃进行功能化处理。
Zotero 插件