Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion
Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion
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DOI：
10.1371/journal.pone.0097440
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发表时间：
2014-05-12
期刊：
影响因子：
3.7
通讯作者：
Mueller, Joachim D.
中科院分区：
文献类型：
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作者：
Hur, Kwang-Ho;Macdonald, Patrick J.;Mueller, Joachim D.
荧光标记蛋白质的亮度为识别活细胞中的蛋白质相互作用提供了一个很好的标记。然而，对亮度的定量解释取决于对影响荧光标签的信号波动的过程的详细了解。在这里，我们关注光漂白对细胞亮度测量的累积影响。在有限的细胞体积内的光漂白会导致荧光标记蛋白质的数量随着时间的推移而耗尽。光耗尽的过程减少了荧光信号，这对亮度数据的分析造成了偏差。我们的数据表明，即使信号的微小减少也会在数据分析中引入显著的偏差。我们开发了一个量化偏差的模型，并引入了一种分析方法，通过对哺乳动物和酵母细胞的实验验证，该方法可以准确地确定光耗尽存在时的亮度。此外，荧光蛋白EGFP的光耗尽实验揭示了光转换过程的存在，这导致EGFP蛋白的亮度显著降低。我们还确定了可以安全地忽略EGFP的光转换对亮度实验的影响的条件。
The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored.