Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy.
Biosens Bioelectron
; 221: 114917, 2023 Feb 01.
Article
em En
| MEDLINE
| ID: mdl-36450170
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.
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Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Microscopia
Limite:
Humans
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article