Real-Time Detection of Intracellular Oxidative Dynamics during Proton Flash Irradiation
Abstract
Purpose
Ultrahigh dose-rate (FLASH) radiotherapy delivers radiation within millisecond timescales, yet direct measurement of radiolytic oxidative processes during beam delivery remains technically challenging. This study aimed to develop and apply a real-time optical detection platform to characterize intracellular oxidative dynamics during proton FLASH irradiation and to compare these responses with conventional dose-rate delivery.
Methods
A fiber-coupled fluorescence detection system was integrated with a 230 MeV clinical proton beamline to enable continuous optical acquisition during irradiation. Fluorescence signals were recorded at 123 Hz, providing millisecond-scale temporal resolution. Intracellular hydrogen peroxide dynamics were monitored using the boronate-based probe BES-H₂O₂-Ac under either conventional dose-rate irradiation (0.2 Gy/s) or FLASH irradiation (>110 Gy/s). Experiments were performed in normal epithelial BEAS-2B cells and lung carcinoma A549 cells. Fluorescence signals were corrected to quantify relative changes associated with irradiation and cell-specific oxidative responses. Relative fluorescent unit (ΔRFU) is used to quantitatively describe the FLASH effect.
Results
The system successfully resolved distinct oxidative kinetics during the beam-on interval. FLASH irradiation produced a rapid fluorescence increase to the ~70 ms beam-on window, with peak slopes of 602.33 ΔRFU/s, followed by a post-irradiation decay over tens of seconds. In contrast, conventional dose-rate irradiation produced minimal deviations from baseline under identical total doses. Background-corrected signals demonstrated cell-type–specific FLASH signatures: A549 cells exhibited larger beam-on amplitudes (~50 ΔRFU) and slower post-irradiation decay, whereas BEAS-2B cells showed smaller peaks (~20 ΔRFU) with steeper negative slopes across the 0–80 s interval.
Conclusion
Real-time measurements revealed dose-rate– and cell-type–specific oxidative dynamics during proton FLASH irradiation. These findings provided direct experimental evidence that ultrahigh dose-rate delivery alters early oxidative processes and established a quantitative framework for incorporating time-resolved radiochemistry into FLASH-related medical physics modeling.