Ha Taekjip, Tinnefeld Philip
Department of Physics, University of Illinois at Urbana-Champaign, 61801, USA.
Annu Rev Phys Chem. 2012;63:595-617. doi: 10.1146/annurev-physchem-032210-103340. Epub 2012 Jan 30.
Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own "personalities" regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.
单分子荧光光谱和超分辨率显微镜是当前技术革命的重要组成部分，能够以前所未有的清晰度和精度揭示生物化学和细胞过程。对荧光团光物理性质的要求非常严格，这推动了合适探针的选择。这类荧光团并非简单的具有特定颜色和亮度的灯泡，而是在光谱参数、氧化还原性质、大小、水溶性、光稳定性以及其他几个因素方面有其自身的“个性”。在此，我们综述了用于粒子追踪、单分子荧光共振能量转移、化学计量测定和超分辨率成像等应用中的荧光探针（包括有机荧光团和荧光蛋白）的光物理性质。特别值得关注的是Cy5的巯基诱导闪烁现象，这在单分子生物物理研究中曾是个难题，不过后来通过使用Trolox的氧化还原系统得以克服，但由于其可控的光开关特性，这一现象在超分辨率成像中却成了好事。理解光物理性质对于单分子实验的设计和解释至关重要。
