Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.
Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.
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用于分子诊断和医学的单细胞测定：通过双光子荧光寿命成像监测细胞内大分子浓度
DOI：
10.7150/thno.11863
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发表时间：
2015
期刊：
影响因子：
12.4
通讯作者：
Prasad PN
中科院分区：
文献类型：
--
作者：
Pliss A;Peng X;Liu L;Kuzmin A;Wang Y;Qu J;Li Y;Prasad PN
细胞的分子组织是随着细胞的生理过程、病理发展或与药物的相互作用而动态转变的。测量和监测单个细胞中大分子浓度的能力将大大加强对异质群体中细胞过程的研究。在本文中，我们介绍并实验验证了一种生物分析单细胞测定方法，其中在特定的亚细胞区域（如细胞核的结构-功能区室以及核质）中估计大分子的总浓度。我们使用双光子激发荧光寿命成像（FLIM）描述了局部生物分子浓度的定量映射，这些浓度要么与细胞的功能和生理状态有关，要么被治疗药物作用改变。该方法利用荧光团的荧光寿命与其微环境的折射率之间的相关性，而微环境的折射率是由于大分子（主要是蛋白质）浓度的变化而变化的。与传统的单光子方法相比，近红外生物透明窗口的双光子激发降低了活细胞的光毒性。利用这种新方法，我们估计核斑区室和核质中的蛋白质平均浓度为~150 mg/ml，核仁中的蛋白质平均浓度为~284 mg/ml。此外，我们还显示了RNA合成药物抑制剂对细胞内蛋白质密度的深远影响。本文提出的方法将在单细胞水平上显著推进治疗学和药物-细胞相互作用的研究，有助于个人分子医学的发展。
Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine.
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影响因子：
4
作者：
Pliss, Artem;Zhao, Lingling;Prasad, Paras N.
通讯作者：
Prasad, Paras N.
DOI：
10.1073/pnas.88.18.8057
发表时间：
1991-09-01
影响因子：
11.1
作者：
SCALETTAR, BA;ABNEY, JR;HACKENBROCK, CR
通讯作者：
HACKENBROCK, CR
影响因子：
64.5
作者：
Spector, David L
通讯作者：
Spector, David L
影响因子：
64.8
作者：
Phair, RD;Misteli, T
通讯作者：
Misteli, T
影响因子：
48
作者：
Choi, Wonshik;Fang-Yen, Christopher;Feld, Michael S.
通讯作者：
Feld, Michael S.