Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.
Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.
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荧光寿命和荧光显微镜的新颖应用使得能够定量了解植物细胞中的亚细胞动力学。
DOI：
10.1371/journal.pone.0005716
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发表时间：
2009-05-27
期刊：
影响因子：
3.7
通讯作者：
Harter K
中科院分区：
文献类型：
--
作者：
Elgass K;Caesar K;Schleifenbaum F;Stierhof YD;Meixner AJ;Harter K
为了更深入地了解活细胞中的分子过程和机制，需要在亚细胞分辨率下进行定量输出的光学和光谱技术。这些技术是在细胞和亚细胞水平上实现预测生物学的先决条件。然而，尽管这些技术建立在物理科学中，但很少应用于植物科学中的细胞生物学。在这里，我们提出了一种结合应用单色团荧光寿命显微镜和波长选择荧光显微镜来分析具有高空间和时间分辨率的油菜素类固醇不敏感1受体(BRI1-GFP)的GFP融合在组织环境中活的拟南芥细胞中的功能。在体内，我们发现油菜素内酯诱导的细胞壁快速扩张和BR调节的细胞膜BRI1-GFP荧光寿命的快速变化。细胞壁的扩张和荧光寿命的变化都反映了BR诱导的早期和BRI1依赖的生理或信号过程。我们的实验还显示了以GFP融合蛋白为探针的单色团荧光寿命显微镜在体内监测生物化学和生物物理亚细胞环境的潜力。单色团荧光寿命显微镜与波长特定的荧光显微镜相结合，为高分辨率的体内动态和定量分析细胞过程开辟了新的前沿，这是纯成像技术或透射电子显微镜无法解决的。
Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences. Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes. One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.
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影响因子：
64.5
作者：
Li, J;Wen, JQ;Walker, JC
通讯作者：
Walker, JC
DOI：
10.1073/pnas.97.7.3718
发表时间：
2000-03-28
影响因子：
11.1
作者：
Cutler, SR;Ehrhardt, DW;Somerville, CR
通讯作者：
Somerville, CR
影响因子：
3.4
作者：
Hink, Mark A.;Shah, Khalid;Visser, Antonie J. W. G.
通讯作者：
Visser, Antonie J. W. G.
影响因子：
3.4
作者：
Böse, G;Schwille, P;Lamparter, T
通讯作者：
Lamparter, T
影响因子：
3
作者：
Martini, Joerg;Schmied, Katja;Merkle, Thomas
通讯作者：
Merkle, Thomas