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Measuring DNA content in live cells by fluorescence microscopy

机译：通过荧光显微镜测量活细胞中的DNA含量

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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. 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. 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.
机译：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，随着时间的流逝同时测量细胞中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含量的能力将使细胞阶段和倍性与各种成像指导分析相结合。在这里，我们描述了一种通过荧光显微镜测量活细胞中DNA含量的广泛适用的无毒方法。这种方法依赖于在任何活细胞成像实验结束时将活细胞膜可渗透的DNA荧光团（例如Hoechst 33342）引入细胞培养基中，并测量每个细胞的整合核荧光以定量DNA含量。重要的是，我们的方法通过战略性地将染料添加到培养物中，从而克服了通常由活细胞染料引起的毒性和对DNA损伤的诱导;使未受干扰的细胞在定量DNA含量之前可以在任何时间间隔内成像。我们根据经验评估方法的性能，并讨论可以使用此技术实现的适应方法。与表达组蛋白2B-GFP融合蛋白（H2B-GFP）的细胞一起提出，我们证明了该方法如何使染色体分离错误在细胞通过细胞分裂过程中得以追踪，随后被鉴定为二倍体或多倍体。我们还描述并提供了一种自动的Matlab衍生算法，该算法可测量每个细胞中的整合核荧光，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。通过并行流式细胞术研究验证了该算法对DNA含量的准确评估。该方法允许以高通量方式将单细胞动力学检查与细胞阶段和倍性相关联。该方法适用于任何配备稳定光源和台式培养箱或封闭活细胞培养箱的标准落射荧光显微镜。总体而言，我们预计该方法将允许对涉及细胞周期进程（例如检查点激活，DNA复制和细胞分裂）的细胞过程进行高分辨率的微观分析。

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