Haas Beth L, Matson Jyl S, DiRita Victor J, Biteen Julie S
Department of Chemistry, University of Michigan, Ann Arbor, MI 48019, USA.
Department of Medical Microbiology and Immunology, University of Toledo, Toledo, OH 43606, USA.
Molecules. 2014 Aug 13;19(8):12116-49. doi: 10.3390/molecules190812116.
Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.
单分子荧光显微镜能够在活细胞内进行生物学研究，以实现毫秒级和纳米级的分辨率。尽管基于单分子的方法越来越容易被非专业人员掌握，但优化新的单分子实验可能具有挑战性，特别是当超分辨率成像和跟踪应用于活细胞时。在这篇综述中，我们总结了活细胞单分子显微镜的常见障碍，并描述了我们开发和应用的方法，以克服活细菌中的这些挑战。我们研究了荧光团和标记方案的选择、实现单分子荧光水平的方法、维持细胞活力的考虑因素，以及在存在噪声和样品漂移的情况下检测单分子信号的策略。我们还讨论了分析单分子轨迹的方法，以及细菌细胞的有限大小和细菌膜的曲率所带来的挑战。
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