Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging
Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging
复制标题
DOI：
10.1016/j.bpj.2008.11.061
复制
发表时间：
2009-03-18
影响因子：
3.4
通讯作者：
Blanchard, Scott C.
中科院分区：
文献类型：
--
作者：
Dave, Richa;Terry, Daniel S.;Blanchard, Scott C.
基于荧光的研究中常见的有机荧光团存在不需要的光物理特性，包括闪烁和光漂白，这限制了它们的整体实验性能。控制此类过程的方法对于单分子荧光和荧光共振能量转移成像尤其重要，其中不间断、稳定的荧光至关重要。已在染料标记的 DNA 和 RNA 系统上进行了基于荧光和 FRET 的测定，以量化小分子溶液添加剂对菁和 Alexa 的荧光和 FRET 行为的影响。荧光团。对超过 200,000 个单个分子的荧光和 FRET 轨迹的详细停留时间分析表明，之前被鉴定为三重态猝灭剂的两种化合物，环辛四烯和 Trolox，以及 4-硝基苯甲醇，可以有效地减弱眨眼、光漂白，并以浓度依赖性和环境依赖性方式影响光复活速率。在所检查的两个生化系统中，独特的化合物混合物被证明对于成像性能而言是最佳的。通过同时提供对多个光物理动力学参数的最快速和直接的访问，smFRET 成像为旨在发现新化合物及其有效组合的未来研究提供了强大的途径。这些努力最终可能有助于根据每个特定的实验需求调整有机染料分子的性能。
Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa. fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.