National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 East Paul Dirac Drive, Tallahassee, 32304, Florida, USA.
Phys Chem Chem Phys. 2013 Nov 21;15(43):18771-83. doi: 10.1039/c3cp53719f.
Single molecule localization-based optical nanoscopy was introduced in 2006, surpassing traditional diffraction-limited resolutions by an order of magnitude. Seven years later, this superresolution technique is continuing to follow a trend of increasing popularity and pervasiveness, with the proof-of-concept work long finished and commercial implementations now available. However one important aspect that tends to become lost in translation is the importance of proper sample preparation, with very few resources addressing the considerations that must be made when preparing samples for imaging with single molecule level sensitivity. Presented here is a an in-depth analysis of all aspects of sample preparation for single molecule superresolution, including both live and fixed cell preparation, choice of fluorophore, fixation and staining techniques, and imaging buffer considerations.
基于单分子定位的光学纳米显微镜于 2006 年问世，其分辨率超越了传统的衍射极限，提高了一个数量级。七年后，这种超分辨率技术的普及度仍在持续提高，其概念验证工作早已完成，商业化应用也已实现。然而，在翻译过程中，一个容易被忽视的重要方面是对样品制备的重视，几乎没有资源专门讨论在以单分子灵敏度成像时，必须考虑的样品制备注意事项。本文深入分析了单分子超分辨率成像的各个方面的样品制备，包括活细胞和固定细胞的制备、荧光染料的选择、固定和染色技术以及成像缓冲液的考虑因素。
