Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy

Bauer, N.; Maisuls, I.; Pereira da Graça, A.; Reinhardt, D.; Erapaneedi, R.; Kirschnick, N.; Schäfers, M.; Grashoff, C.; Landfester, K.; Vestweber, D.; Strassert, C. A.; Kiefer, F.

Research article (journal)

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.

Details zur Publikation

Publisher:
Release year: 2023
Publishing company: Elsevier Ltd
Language in which the publication is written: English
Link to the full text: https://api.elsevier.com/content/abstract/scopus_id/85142708231