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首页> 外文会议>SPIE Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues >Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation
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Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

机译：使用rogfp和从一个波长激励获得的荧光光谱测定体内氧化还原电位

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摘要

The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplastlocated Grx 1 -roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.
机译：通过使用绿色荧光蛋白（GFP）及其变体作为特异性细胞标记物，促进了诸如信号转导，DNA复制，碳代谢和衰老等的诸如信号转导，DNA复制，碳代谢和衰老等的分子过程的分析。许多细胞生物过程伴有细胞内氧化还原潜力的变化。为了监测氧化还原电位，产生GFP（RogFP）的氧化还原敏感突变体，其响应于其周围介质的氧化还原状态的变化而显示其光学性质的变化。对于生活系统中的定量分析，必须了解在体外rogfp的光学性质。因此，我们在暴露于不同氧化还原电位的纯化的rogfp上施加光谱分辨的荧光光谱，以确定罗格布的吸收和发射光谱的变化。基于这些体外发现，我们利用一个波长激励来使用Rogfp进行细胞生物过程的体内分析的新方法。我们通过研究表达拟南芥细胞的叶绿体絮凝的GRX 1 -ROGFP2作为动态移动细胞内隔室的实施例，证明了这种技术的能力。这对于到目前为止建立的双波长激励技术是不可能的，这妨碍了由于两次测量和所研究区域的后续位移之间的时间延迟而进行高度移动样品的定量分析。

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