Feasibility of ESR measurements in living zebrafish embryos following conventional and ultra-high dose rate irradiation

Objective: The FLASH effect has been reported to reduce normal tissue toxicity while maintaining tumour control, yet the underlying mechanisms remain incompletely understood. Radiochemical processes, including oxygen-dependent radical reactions, have been proposed as potential contributors. To support future investigations of these processes, this feasibility and method-development study aimed to establish an electron spin resonance (ESR) workflow for detecting radical signals in living zebrafish embryos (ZFE) following conventional dose rate (CDR) and ultra-high dose rate (UHDR) electron irradiation, and to assess its compatibility with subsequent biological endpoint assessment. Approach: A nitroxide spin probe was used to quantify radical formation in irradiated E3 medium and living ZFE exposed to both radiation qualities under two different oxygenation conditions. In parallel, survival, hatching, body length, eye diameter, pericardial edema, and spine curvature were assessed to evaluate biological radiation effects. Potential influences of spin probe exposure and the complete ESR workflow on ZFE development were analysed separately. Main results: ESR measurements in E3 medium reproduced previously reported dose-rate- and oxygen-dependent differences in radical formation. In ZFE, radical signals could be reliably detected under the applied experimental conditions, demonstrating the feasibility of ESR measurements in living embryos. Exploratory comparisons revealed no statistically significant differences in ZFE ESR signals between CDR and UHDR irradiation. Neither spin probe exposure nor ESR measurements resulted in significant effects on survival, growth, or structural abnormalities. Under hypoxic conditions, significant healthy tissue sparing of UHDR relative to CDR irradiation was observed for body length and eye diameter, whereas no significant effect was detected under physioxic conditions. Significance: These findings demonstrate the feasibility of ESR measurements in living ZFE and support their application in future studies investigating the relationship between radical formation and biological radiation effects.