Benchtop X-band electron paramagnetic resonance detection of melanin and Nitroxyl spin probe in zebrafish.

EPR spectroscopy is a technique that provides direct information about free radicals in biological systems. So far, X-band EPR was seldomly used for in vivo studies as the small resonator size and high power used to detect EPR signals were unsuitable for living organisms. Here, we report new solutions which lift some limitations and make X-band EPR suitable for an in vivo detection of free radicals in zebrafish - a small laboratory animal that is often used as a model for various studies related to free radicals. We designed specially-shaped glass and quartz capillaries to ensure the zebrafish's safety during the experiments. The optimal EPR spectrometer parameters, safe for zebrafish embryos and sufficient to obtain EPR spectrum, were 4 scans by 20s, 100G sweep, and 0.8 mW power. Combining the specially-shaped capillary with a multi-harmonic analyzer for the EPR spectrometer allowed increasing the time up to 16 scans by 11s and lowering the power to 0.25 mW. As a proof of principle, we demonstrate the detection of melanin radicals and the 5-DSA spin probe in zebrafish larvae. As fish survive the EPR scans, the possibility of performing multiple measurements of free radicals in living zebrafish offers new tools for studies aiming to understand redox biology and membrane-dependent functions in both health and disease. [Display omitted] • Short exposure to low power X band EPR radiation is safe for zebrafish. • Specially shaped capillary ensures proper positioning of the sample and tuning of EPR. • Glass and quartz capillaries are both effective for the measurement of free radicals. • Melanin radical could be observed in vivo in zebrafish larvae with X band EPR. • Membrane fluidity changes could be observed in vivo in zebrafish larvae with X band EPR. [ABSTRACT FROM AUTHOR]