Author: Yuqi Yang, Fang-Fang Yin ðĻâðŽ
Affiliation: Duke Kunshan University, Medical Physics Graduate Program, Duke Kunshan University ð
Purpose: Reactive oxygen species (ROS) are unstable molecules which play important roles in the cellular process after Flash radiotherapy. The rapid changes in ROS levels during treatment may provide insights into the Flash effect mechanism and immune response. Fluorescent probes have potential for real-time ROS detection in cells and tissues, however, their performance under Flash radiation conditions has not been verified. This study aims to investigate the feasibility of using fluorescent probes for real-time ROS detection during Flash radiotherapy by evaluating their performance under high dose and high dose rate conditions.
Methods: Three fluorescent probes, DCFH-DA, BES-H2O2-Ac and Amplex Red, were selected for general ROS and specific hydrogen peroxide detection. To assess the stability and sensitivity, probes were irradiated with proton beam at 50 Gy/s and 100 Gy total dose. Using a microphotometer, fluorescence power was recorded in real-time with 485nm/530nm excitation light and 515nm/582 emission filters, corresponding to different probes. The relationship between the fluorescence power of each probe and time was plotted. The feasibility of using fluorescence probes was evaluated by assessing the consistency of fluorescence signal changes with expected ROS dynamics.
Results: Flash radiation conditions showed negligible effects on probes stability. Statistical analysis with low standard deviations and non-significant p-values, confirmed no significant differences between irradiated and non-irradiated conditions. Additionally, the maximum fluorescence powers of DCFH-DA, BES- H2O2-Ac, and Amplex Red were 49,683 fW, 26,195 fW, and 249,999 fW, respectively, in the presence of 20 ΞM H2O2. These results demonstrated a quantitative relationship between fluorescence power and H2O2 concentration.
Conclusion: This study verified the feasibility of using fluorescence probes under Flash radiotherapy conditions, as a reliable tool for investigating the ROS dynamics in vitro. These findings provide a foundation for further research into ROS behavior during Flash radiotherapy.