Quantitative real-time analysis of nucleolar stress by coherent phase microscopy
Quantitative real-time analysis of nucleolar stress by coherent phase microscopy
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DOI：
10.1117/1.3042241
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发表时间：
2008-11-01
影响因子：
3.5
通讯作者：
Shtil, Alexander A.
中科院分区：
文献类型：
--
作者：
Tychinsky, Vladimir P.;Kretushev, Alexander V.;Shtil, Alexander A.
我们开发了一种相干相位显微镜(CPM)方法，用于直接显示未固定、未染色的哺乳动物细胞(包括培养细胞和新分离的肿瘤活检组织)，然后进行计算机辅助数据分析。CPM的主要目的是评估光学致密的细胞内结构，如细胞核和核仁的折射特性。特别是，我们专注于使用相位厚度作为光学非均匀生物对象的光程差的等价物来实时定量分析核仁动力学。药物抑制基因转录导致核仁的相厚度在细胞暴露的最初几分钟内急剧减少。此外，细胞内ATP池的急性耗竭、微管的解聚和DNA复制的抑制导致核仁相厚度迅速减少。这些光学效应与电子显微镜记录的核仁成分的分离是平行的。因此，CPM检测核仁动力学的早期变化，特别是核仁分离，作为细胞对细胞毒应激反应的一部分，无论核仁是否是毒素的主要靶标。CPM适用于监测和定量分析活的哺乳动物细胞中的“核仁应力”。(C)2008年光学仪器工程师学会。[DOI：10.1117/1.3042241]
We develop a method of coherent phase microscopy (CPM) for direct visualization of nonfixed, nonstained mammalian cells (both cultured cells and freshly isolated tumor biopsies) followed by computer-assisted data analysis. The major purpose of CPM is to evaluate the refractive properties of optically dense intracellular structures such as the nucleus and the nucleoli. In particular, we focus on quantitative real-time analysis of the nucleolar dynamics using phase thickness as an equivalent of optical path difference for optically non-homogenous biological objects. Pharmacological inhibition of gene transcription leads to a dramatic decrease of the phase thickness of the nucleoli within the initial minutes of cell exposure. Furthermore, the acute depletion of intracellular ATP pool, depolymerization of microtubules and inhibition of DNA replication resulted in a rapid decrease of the nucleolar phase thickness. These optical effects were paralleled by segregation of nucleolar components as documented by electron microscopy. Thus, CPM detects early changes of nucleolar dynamics, in particular, the nucleolar segregation as part of general cellular response to cytotoxic stress, regardless of whether the nucleolus is or is not the primary target of the toxin. CPM is applicable for monitoring and quantitative analysis of the "nucleolar stress" in living mammalian cells. (c) 2008 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3042241]