Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

S. Wierer; K. Elgass; S. Bieker; U. Zentgraf; A. J. Meixner; F. Schleifenbaum

doi:10.1117/12.873753

22 February 2011 Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

S. Wierer, K. Elgass, S. Bieker, U. Zentgraf, A. J. Meixner, F. Schleifenbaum

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Proceedings Volume 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX; 790211 (2011) https://doi.org/10.1117/12.873753
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

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 chloroplast-located Grx1-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.

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S. Wierer, K. Elgass, S. Bieker, U. Zentgraf, A. J. Meixner, and F. Schleifenbaum "Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation", Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 790211 (22 February 2011); https://doi.org/10.1117/12.873753

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KEYWORDS

Luminescence

Chromophores

Green fluorescent protein

In vivo imaging

In vitro testing

Optical properties

Biological research

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