Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy
Nadine Bauer, Iván Maisuls, Abel Pereira da Graça, Dirk Reinhardt, Raghu Erapaneedi, Nils Kirschnick, Michael Schäfers, Carsten Grashoff, Katharina Landfester, Dietmar Vestweber, Cristian A. Strassert, Friedemann Kiefer
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.