自动化透射电子显微镜图像分析：染色质背景下辐射诱导 DNA 损伤的纳米尺度评估。

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

机构信息

Department of Radiation Oncology, Saarland University Medical Center, Kirrbergerstr, Building 6.5, 66421 Homburg, Saar, Germany.

出版信息

Cells. 2023 Oct 10;12(20):2427. doi: 10.3390/cells12202427.

DOI:10.3390/cells12202427

PMID:37887271

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10605235/

Abstract

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

摘要

背景

具有高线性能量转移（LET）的重离子辐照（IR）的特点是具有独特的深度剂量分布和增加的生物学效应。在高 LET 射线照射后，沿着粒子轨迹的局部能量沉积会导致 DNA 损伤簇集，从而产生低电子密度域（LEDD）。为了研究 DNA 修复和染色质重塑的时空动力学，我们建立了透射电子显微镜图像的自动分析。

方法

用高 LET 碳离子或低 LET 光子照射人成纤维细胞。在辐照后 0.1 h、0.5 h、5 h 和 24 h，使用透射电子显微镜在间期核中可视化纳米颗粒标记的修复因子（53BP1、pKu70、pKu80、DNA-PKcs），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，以评估低 LET 与高 LET IR 后的 DNA 损伤模式。

结果

低 LET 射线照射会在整个细胞核中诱导单个 DNA 损伤，并且大多数 DNA 双链断裂（DSB）都可以有效地重新连接，没有可见的染色质去浓缩。高 LET 射线照射会诱导沿着粒子轨迹集中的 DNA 损伤簇集，导致有界的 LEDD。自动图像分析用于确定不同大小纳米颗粒的精确数量、它们彼此之间的距离以及它们在显微照片中的精确位置（基于大小、形状和密度）。染色质密度是从灰度特征确定的，纳米颗粒自动分配给常染色质或异染色质。使用自动分割来描绘高 LET 诱导的 LEDD，并确定纳米颗粒在分割的 LEDD 中的空间分布。