Imaging and Spectroscopy of Radiation Chemistry in FLASH Radiotherapy to Inform Clinical Translation

FLASH radiotherapy (RT) is a novel and transformative approach that delivers ultra-high dose rate radiation (>40 Gy/s), offering the potential to significantly reduce normal tissue toxicity while maintaining tumor control. Despite compelling preclinical and early clinical data, the mechanisms underlying the FLASH effect remain poorly understood, hindering its widespread clinical adoption. The central hypothesis of this proposal is that FLASH RT alters the radiochemical landscape during the first microseconds to milliseconds post-irradiation, leading to a shift in reactive species (RS) composition and dynamics that underlies the observed tissue sparing. Specifically, we propose that FLASH RT increases the yield of aqueous electrons (e⁻aq) and suppresses downstream oxidative stress through altered spur kinetics and radical recombination, resulting in reduced DNA damage and long-term tissue toxicity. The overall objective of this proposal is to elucidate the mechanistic basis of the FLASH effect by directly measuring RS production and biological responses under clinically relevant irradiation conditions. To achieve this, we will develop three complementary platforms: (1) time-resolved absorption spectroscopy for label-free quantification of RS with sub-microsecond resolution, (2) live-cell fluorescence imaging and super-resolution microscopy to map nanoscale RS dynamics and DNA damage repair, and (3) in vivo absorption spectroscopy (ALIVE) to measure RS kinetics in irradiated tissues and correlate them with acute and chronic toxicity endpoints. While our in vitro platforms isolate early radiochemical mechanisms at the nanoscale, live animal models are required to determine how these events propagate through intact tissue architecture. This in vivo context is essential to definitively link initial RS dynamics with macroscopic, long-term physiological outcomes. This work is highly innovative in integrating custom-built optical systems with FLASH-capable beamlines to enable direct, real-time measurement of RS under ultra-high dose rates. Unlike conventional assays relying on probes or indirect surrogates, our approach allows for label-free detection of short-lived species such as e⁻aq, providing unprecedented insight into the earliest radiochemical events that drive biological outcomes. By systematically varying beam parameters, including dose per pulse, mean dose rate, and pulse structure, we will generate a mechanistic map linking physical delivery characteristics to RS production and biological response. The clinical impact of this work is substantial. By identifying the radiochemical and biological signatures that define the FLASH effect, we will enable rational optimization of treatment protocols tailored to specific tissue types and clinical indications. This will support the development of predictive biomarkers for FLASH responsiveness, guide patient selection, and inform dose fractionation strategies. Ultimately, this research will accelerate the safe and effective translation of FLASH RT into routine clinical practice, improving therapeutic outcomes, reducing treatment-related morbidity, and enhancing quality of life for cancer patients. Project Number: 1R01CA315651-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Anna Karin Gustavsson (+1 co-PI) | Institution: RICE UNIVERSITY, HOUSTON, TX | Award Amount: $369,394 | Activity Code: R01 | Study Section: Radiation Therapeutics and Biology Study Section[RTB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11425869