ABSTRACT Significant improvement in the effectiveness of radiation therapy (RT) now seems possible because of recent exciting research results using ultra-high dose rates (UHDR), indicating that normal tissue damage can be reduced (the `FLASH' effect), compared to conventional radiation for the same total dose. FLASH RT delivery has shown reduced morphological and functional damage to normal tissues such as brain, colon, lung, and skin. Although significant research remains, early indications are that the normal tissue sparing effect may as high 50% at some dose levels. If proven in translation, this effect would be the most significant improvement in RT therapeutic ratio since the advent of treatment planning. While intriguing mechanisms for this result have been postulated, they remain only well-reasoned speculations because of a lack of direct in vivo data on the physico- chemical mechanisms including oxygen depletion and free radical species alterations. Dartmouth has created the first reversible MeV FLASH beam on a clinically commissioned linac with >100 Gy/s at the patient treatment bed, and prototyped an open-source treatment planning system, expanding research access. Additionally, the team has invented unique technological capabilities to directly measure the highest dose rates and in vivo tissue oxygen transients. The key technological barriers solved in the proposed research with preliminary data are: 1) demonstration of conversion to a UHDR irradiator within a clinically commissioned linac, 2) verification of per- pulse and per-fraction dose rates, 3) direct in vivo observation of oxygen transients, 4) direct measurements of free radical species changes in vitro, and 5) access to functional tissue assays and genetic and proteomic assays. Single-pulse and single-fraction dose rates will be quantified by high frame rate imaging. Additionally, the in vivo oxygen changes will be quantified by two independent methods for co-validation, including electron paramagnetic resonance oximetry and optical luminescence oximetry. Free radical species changes produced from transient hypoxia will also be assessed through systematic in vitro analyses, and the potential linkages to functional, proteomic and DNA damage examined. The FLASH beam conditions that minimize normal tissue damage for a fixed dose will be established with this baseline data. The work is pre-clinical but can be readily adapted to ongoing NIH sponsored spontaneous canine cancer studies has relevance to future large animal and first-in-human translation. Multiple Dartmouth centers partnered to initiate and support this FLASH program. Taken altogether, this bioengineering research project will advance the state of the art in high dose rate radiation therapy using tools that have been uniquely developed by our 3 research groups, and the project results will build the basic science needed to support proposed human translation of this ground-breaking field. The team has leading expertis...