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Genetically encoded FRET probes for direct mapping and quantification of intracellular oxygenation level via fluorescence lifetime imaging
Author(s): Alessio Andreoni; Rozhin Penjweini; Branden Roarke; Marie-Paule Strub; Dan L. Sackett; Jay R. Knutson
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Paper Abstract

Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration is used for the evaluation of many diseases (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration ([O2]) in living cells, tissues and organisms is challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor [O2] in the intracellular environment. We fused Myoglobin (Myo), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair, Myo-mCherry. The changes in the spectral properties of Myoglobin upon oxygen binding result in changes of the FRETdepleted emission intensity of mCherry, and this effect is detected by monitoring the fluorescence lifetime of the probe. We present here the preparation and characterization of a series of Myo-mCherry variants and mutants that show the versatility of our protein-based approach: the dynamic range of the sensor is tunable and adaptable to different [O2] ranges, as they occur in vitro in different cell lines, the probe is also easily targeted to subcellular compartments. The use of fluorescence overcomes the most common issues of data collection speed and spatial resolution encountered by currently available methods for O2-monitoring. By using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we can map the oxygenation level of cells in vitro, providing a quantitative assessment of [O2].

Paper Details

Date Published: 22 February 2019
PDF: 10 pages
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820O (22 February 2019); doi: 10.1117/12.2510646
Show Author Affiliations
Alessio Andreoni, National Institutes of Health (United States)
Rozhin Penjweini, National Institutes of Health (United States)
Branden Roarke, National Institutes of Health (United States)
Marie-Paule Strub, National Institutes of Health (United States)
Dan L. Sackett, National Institutes of Health (United States)
Jay R. Knutson, National Institutes of Health (United States)


Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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