A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci.
A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci.
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DOI：
10.1038/s42003-022-03585-5
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发表时间：
2022-07-14
影响因子：
5.9
通讯作者：
中科院分区：
文献类型：
--
作者：
双链断裂标记的免疫荧光标记，如γ-H_2AX和其他双链修复蛋白，是理解辐射后生物学后果的有力工具。然而，尽管这项技术被广泛使用，但在使用显微镜计数时，与其分辨和可靠地推断病灶数量的能力相关的还有许多不确定因素。我们提出了一种新的工具，用于模拟辐射诱导的焦点，以评估电子免疫荧光图像中的显微镜性能。利用蒙特卡罗轨道结构模拟产生了DSB分布的模拟。对于每个DSB分布，模拟相应的DNA修复过程，并在几个时间点记录未修复的DSB。生成了DSB和(γ-H_2AX)荧光标记的相应显微图像，并对不同显微镜、辐射类型和剂量进行了比较。在大多数测试场景中，都发现了统计上的显著差异。这些不一致被传播到修复动力学，在那里有辐射类型之间的感知变化。这些变化并不能反映潜在的修复率，而是由于病灶计数的不一致造成的。我们的结论是，在分析DNA损伤标记的图像时，必须考虑这些潜在的不确定性，以确保观察到的差异是真实的，而不是由非系统的错误计算造成的。PyFoci是一种模拟荧光标记的DNA双链断裂标记蛋白质焦点的分布的工具，可以在不同的辐射类型、剂量和显微镜设置下估计误计数。
Immunofluorescent tagging of DNA double-strand break (DSB) markers, such as γ-H2AX and other DSB repair proteins, are powerful tools in understanding biological consequences following irradiation. However, whilst the technique is widespread, there are many uncertainties related to its ability to resolve and reliably deduce the number of foci when counting using microscopy. We present a new tool for simulating radiation-induced foci in order to evaluate microscope performance within in silico immunofluorescent images. Simulations of the DSB distributions were generated using Monte Carlo track-structure simulation. For each DSB distribution, a corresponding DNA repair process was modelled and the un-repaired DSBs were recorded at several time points. Corresponding microscopy images for both a DSB and (γ-H2AX) fluorescent marker were generated and compared for different microscopes, radiation types and doses. Statistically significant differences in miscounting were found across most of the tested scenarios. These inconsistencies were propagated through to repair kinetics where there was a perceived change between radiation-types. These changes did not reflect the underlying repair rate and were caused by inconsistencies in foci counting. We conclude that these underlying uncertainties must be considered when analysing images of DNA damage markers to ensure differences observed are real and are not caused by non-systematic miscounting. PyFoci is a tool that simulates distributions of fluorescently labeled DNA double-strand break marker protein foci and allows the estimation of miscounting under different radiation types, doses and microscopy settings.
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影响因子：
4.3
作者：
Ingram SP;Henthorn NT;Warmenhoven JW;Kirkby NF;Mackay RI;Kirkby KJ;Merchant MJ
通讯作者：
Merchant MJ
影响因子：
2.3
作者：
Ballarini F
通讯作者：
Ballarini F
DOI：
10.1186/1748-717x-8-155
发表时间：
2013-06-26
期刊：
Radiation oncology (London, England)
影响因子：
--
作者：
Li P;Du CR;Xu WC;Shi ZL;Zhang Q;Li ZB;Fu S
通讯作者：
Fu S
影响因子：
--
作者：
Hagiwara Y;Niimi A;Isono M;Yamauchi M;Yasuhara T;Limsirichaikul S;Oike T;Sato H;Held KD;Nakano T;Shibata A
通讯作者：
Shibata A
DOI：
10.1016/j.ijrobp.2009.09.040
发表时间：
2010-03-01
影响因子：
7
作者：
Bentzen, Soren M.;Constine, Louis S.;Deasy, Joseph O.;Eisbruch, Avi;Jackson, Andrew;Marks, Lawrence B.;Ten Haken, Randall K.;Yorke, Ellen D.
通讯作者：
Yorke, Ellen D.