背景:具有高线性能量转移(LET)的重离子辐照(IR)的特征在于独特的深度剂量分布和增加的生物有效性。在高LETIR之后,沿粒子轨迹的局部能量沉积诱导成簇的DNA损伤,导致低电子密度域(LED)。探讨DNA修复和染色质重塑的时空动态,我们建立了透射电子显微照片的自动图像分析。
方法:用高LET碳离子或低LET光子照射人成纤维细胞。0.1h时,0.5h,5h,和24小时后IR,纳米颗粒标记的修复因子(53BP1,pKu70,pKu80,DNA-PKcs)使用透射电子显微镜在相间核中可视化,以监测染色质超微结构中DNA损伤的形成和修复。使用基于AI的软件工具,建立了先进的图像分析技术来评估低LET与高LETIR后的DNA损伤模式。
结果:低LETIR诱导整个细胞核的单个DNA损伤,大多数DNA双链断裂(DSB)有效地重新连接,没有可见的染色质去缩合。高LETIR诱导的聚集DNA损伤集中在粒子轨迹上,导致受限制的LED。自动图像分析用于确定不同大小的纳米粒子的确切数量,彼此的距离,以及它们在显微照片中的精确位置(基于尺寸,形状,和密度)。根据灰度特征确定染色质密度,纳米粒子自动分配给常染色质或异染色质。使用自动分割来描绘高LETIR诱导的LED,并确定了纳米粒子相对于分段LED的空间分布。
结论:我们的图像分析结果表明,高LETIR诱导沿着粒子轨迹的染色质松弛,实现对连续DNA损伤的关键修复。暴露于不同的辐射质量后,染色质超微结构中纳米颗粒标记的DNA修复蛋白的自动图像分析能够精确表征特定的DNA损伤模式。
Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs.
Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR.
Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined.
The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.