In cellulo Evaluation of Phototransformation Quantum Yields in Fluorescent Proteins Used As Markers for Single-Molecule Localization Microscopy
In cellulo Evaluation of Phototransformation Quantum Yields in Fluorescent Proteins Used As Markers for Single-Molecule Localization Microscopy
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DOI：
10.1371/journal.pone.0098362
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发表时间：
2014-06-10
期刊：
影响因子：
3.7
通讯作者：
Bourgeois, Dominique
中科院分区：
文献类型：
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作者：
Avilov, Sergiy;Berardozzi, Romain;Bourgeois, Dominique
生物样品的单分子定位显微镜需要精确了解所用的荧光标记。可光转化荧光蛋白的光活化、光闪烁和光漂白影响（荧光）光活化定位显微术（（F）-PALM）中使用的数据采集和数据处理策略，特别是用于可靠的分子计数。由于这些参数可能取决于当地环境，因此应在生物相关实验条件下在细胞中进行测量。在这里，我们测量了典型的（F）-PALM实验中固定的哺乳动物细胞中Dendra 2融合到肌动蛋白的光转化量子产率。为此，我们开发了一种基于Lee等人提出的聚类优化过程的数据处理策略（PNAS 109，17436-17441，2012）。使用模拟，我们估计的范围内的实验参数（分子密度，分子取向，背景水平，激光功率，帧时间）足以准确测定的光转化产量。在pH 7.4的PBS缓冲液中，在561 nm的光照下，Dendra 2融合到肌动蛋白上的光漂白产率测量为（2.5 +/- 0.4）x 10（-5），而Blinking-off产率和热激活Blinking-on速率分别测量为（2.3 +/- 0.2）x 10（-5）和11.7 +/- 0.5 s（-1）。这些光转化产率不同于在聚乙烯醇（PVA）中测量的那些，并且受到抗褪色剂1，4-二氮杂双环[2.2.2]辛烷（DABCO）的强烈影响。在DABCO存在下，光漂白产率降低2倍，眨眼率降低3倍以上，眨眼率增加2倍。因此，DABCO大大提高了Dendra 2在固定的哺乳动物细胞中的光稳定性。这些发现与（F）-PALM实验条件下基于氧化还原的漂白和眨眼机制一致。最后，在405 nm光照下，Dendra 2的绿色到红色光转换量子产率估计为（1.4 +/- 0.6）x 10（-5）。
Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436-17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5 +/- 0.4) x 10(-5), whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3 +/- 0.2) x 10(-5) and 11.7 +/- 0.5 s(-1), respectively. These phototransformation yields differed from those measured in polyvinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2] octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4 +/- 0.6) x 10(-5) in cellulo under 405 nm illumination.