Background Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. Results Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. Conclusions This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

中文翻译：

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通过荧光显微镜测量活细胞中的 DNA 含量。

背景 活细胞荧光显微镜 (LCFM) 是用于实时研究细胞动力学的强大工具。然而，随着时间的推移同时测量细胞中 DNA 含量的能力仍然受到染料相关毒性的挑战。通过 LCFM 测量单个细胞中 DNA 含量的能力将允许细胞阶段和倍性与各种成像定向分析相结合。在这里，我们描述了一种广泛适用的无毒方法，用于通过荧光显微镜测量活细胞中的 DNA 含量。该方法依赖于在任何活细胞成像实验结束时将活细胞膜渗透 DNA 荧光团（例如 Hoechst 33342）引入细胞培养基，并测量每个细胞的综合核荧光以量化 DNA 含量。重要的，我们的方法通过将染料添加到培养物中的战略时机克服了通常由活细胞染料引起的毒性和 DNA 损伤诱导；允许在量化其 DNA 含量之前对未受干扰的细胞进行任何时间间隔的成像。我们根据经验评估我们方法的性能，并讨论可以使用这种技术实现的适应。结果 结合表达组蛋白 2B-GFP 融合蛋白 (H2B-GFP) 的细胞，我们展示了这种方法如何在细胞分裂过程中追踪染色体分离错误，这些细胞分裂后来被确定为二倍体或多倍体。我们还描述并提供了一种自动化的 Matlab 衍生算法，该算法测量每个细胞中的综合核荧光，然后将这些测量结果绘制成每个成像帧的细胞周期直方图。该算法对 DNA 含量的准确评估通过平行流式细胞术研究得到验证。结论该方法允许以高通量方式检查单细胞动力学与细胞阶段和倍性相关。该方法适用于任何配备稳定照明源和台顶培养箱或封闭式活细胞培养室的标准落射荧光显微镜。总的来说，我们预计这种方法将允许对涉及细胞周期进程的细胞过程进行高分辨率显微镜分析，例如检查点激活，