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Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy.
- Source :
-
Biosensors & Bioelectronics . Feb2023, Vol. 221, pN.PAG-N.PAG. 1p. - Publication Year :
- 2023
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Abstract
- Hypoxia is an essential regulator of cell metabolism, affects cell migration and angiogenesis during development and contributes to a wide range of pathological conditions. Multiple techniques to assess hypoxia through oxygen-imaging have been developed. However, significant limitations include low spatiotemporal resolution, limited tissue penetration of exogenous probes and non-dynamic signals due to irreversible probe-chemistry. First genetically-encoded reporters only partly overcame these limitations as the green and red fluorescent proteins (GFP/RFP) families require molecular oxygen for fluorescence. For the herein presented ratiometric and FRET-FLIM reporters dUnORS and dUnOFLS, we exploited oxygen-dependent maturation in combination with the hypoxia-tolerant fluorescent-protein UnaG. For ratiometric measurements, UnaG was fused to the orange large Stokes Shift protein CyOFP1, allowing excitation with a single light-source, while fusion of UnaG with mOrange2 allowed FRET-FLIM analysis. Imaging live or fixed cultured cells for calibration, we applied both reporters in spheroid and tumor transplantation-models and obtained graded information on oxygen-availability at cellular resolution, establishing these sensors as promising tools for visualizing oxygen-gradients in-vivo. • Bipartite cellular hypoxia sensors of O 2 -tolerant & -sensitive fluorescent proteins. • Genetically encoded reporters for intensity- or FRET /FLIM-based measurements. • Graded live-cell microscopic oxygen-imaging in vitro using dUnORS and dUnOFLS. • Optimal working range well within physiological cellular oxygen-concentrations. • Sensors visualized oxygen gradients in intracranial brain tumors ex vivo. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 09565663
- Volume :
- 221
- Database :
- Academic Search Index
- Journal :
- Biosensors & Bioelectronics
- Publication Type :
- Academic Journal
- Accession number :
- 160539497
- Full Text :
- https://doi.org/10.1016/j.bios.2022.114917