Laxman Pooja, Ansari Shirin, Gaus Katharina, Goyette Jesse
European Molecular Biology Laboratory (EMBL) Australia Node in Single Molecule Sciences, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.
Front Chem. 2021 Mar 25;9:641355. doi: 10.3389/fchem.2021.641355. eCollection 2021.
Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.
单分子定位显微镜（SMLM）是一种成像方法，它能够可视化小于光的衍射极限（约200纳米）的结构。这是通过诸如随机光学重建显微镜（STORM）和光激活定位显微镜（PALM）等技术实现的。获得单分子理想成像的很大一部分在于选择合适的荧光标记。蛋白质标记的一个新兴领域是将带有附着荧光染料的非天然氨基酸（UAA）掺入，以实现单个分子的精确定位和可视化。对于这种技术，荧光探针与UAA偶联，并作为标记引入到感兴趣的蛋白质（POI）中。在这里，我们将这种标记方法与其他常用的基于蛋白质的标记方法（如荧光蛋白（FP））或自标记标签（如卤代标签、SNAP标签和CLIP标签）进行对比，并强调在SMLM中UAA与荧光染料位点特异性掺入的优点和缺点。
