Novel In Vivo Research Platform for FLASH Radiation Therapy Radiation therapy (RT) is a major pillar in cancer treatment, but tissue toxicity is a key adverse effect. Normal tissue protection in RT is currently achieved by fractionation and high-precision dose-delivery techniques. Despite major advances in treatment delivery to irradiate tumors and minimize normal tissue involvement (e.g. stereotactic RT), radiation-induced normal tissue toxicities still adversely affect treatment outcome and patients’ quality of life. FLASH-RT exploits a long-overlooked parameter dose rate to enable high curative doses to be delivered to tumors at extremely short time periods by an ultra-high-dose rate (>40Gy/s), while protecting normal tissue. FLASH-RT was found to induce lower normal tissue toxicity than conventional dose-rate irradiation, but to be just as effective in tumor control. Pre-clinical evidence has supported its effectiveness for potential use in clinics. However, the mechanism underlying FLASH’s effect is still unknown and more work is needed for its clinical translation. A few hypotheses related to tissue oxygenation have been proposed to explain the differential radiation response between normal tissue and tumor but have not been validated. Besides elucidating the mechanism, we need thorough studies for clinical translation, such as comparing clinic relevant hypofractionated schemes with FLASH-RT to determine the therapeutic gain level for FLASH-RT. In this respect, biological in vivo studies are especially important. However, a major challenge for FLASH research is that very few systems can deliver ultra-high dose rate irradiation, while none of these systems are equipped with a proper image-guided system. Without an adequate image-guided system, precise in vivo FLASH studies will be difficult and underlying experimental uncertainties can lead to imprecise research conclusion. We believe that a small animal image-guided FLASH (SAIG-FLASH) research platform will enable accurate in vivo FLASH studies to facilitate clinical translation. We plan on developing this research system by 1) modifying a clinical linear accelerator (LINAC) to achieve ultra-high-dose rate irradiation and 2) developing an image-guided research system for FLASH-RT. We will integrate our advanced optical tomography with a CBCT system to provide optimal target localization and assessment capability for in vivo FLASH-RT. A theoretical O2 diffusion model and in vivo O2 measuring system will also be developed as unique research tools to study the FLASH mechanism. FLASH-RT is a paradigm-shift modality that can change the way cancer patients are treated in the future. SAIG-FLASH is especially critical to enable accurate in vivo studies that will facilitate clinical translation.