Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such "prebleaching" can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.

中文翻译：

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校正单分子光漂白测量中的系统偏差

单分子光漂白是一种强大的技术，可用于测量亚分辨率分子复合物中的荧光单位数量，例如与淀粉样蛋白疾病相关的有毒蛋白质寡聚体。但是，在适当放置和聚焦样品之前，可能会发生光漂白。这种“预漂白”可以对较小的低聚物引入强烈的系统偏差。已知预漂白的定量校正是一个不适定的问题，限制了该技术的实用性。在这里，我们提供了一个实验解决方案来提高其可靠性。我们用化学方法构建多聚体标准来估计预漂白概率 B。我们表明 B 可以用作约束来可靠地纠正从标准低聚物的已知分布中获得的统计数据。最后，我们将此方法应用于从人胰岛淀粉样多肽的异质寡聚溶液中获得的数据。我们的结果表明，光漂白会严重歪曲寡聚体分布的估计，因此低丰度单体显示出更高的表观丰度。总之，来自 B > 0.1 的光漂白实验的任何推论都可能不可靠，但我们的方法可用于定量纠正可能的错误。