There is a clear and urgent unmet clinical need for non-invasive, non-ionizing and non-chemo-toxic treatment options for patients with brain tumors. The long-term goal of this proposal is to develop non- thermal ablation (NTA) using phase shift microbubble-aided Magnetic Resonance guided Focused Ultrasound (MRgFUS) into a new non-invasive treatment option that will provide brain cancer patients with increased survival rates and decreased side effects. The overall objective of this application is to (i) develop novel Magnetic Resonance Imaging (MRI) based methods for real-time monitoring and guidance of the non- thermal ablation process; and (ii) adapt a recently developed and validated voxel-wise in vivo MRI to histopathology registration pipeline to brains, and use it to develop and verify imaging biomarkers which can acutely predict long-term tissue non-viability. The rationale for this project is that a means for non-invasive real-time monitoring which directly image the effect of the treatment on the tissue of interest, together with a thorough understanding of non-thermally induced lesion progression and prediction of long-term tissue nonviability are necessary to facilitate safe and timely translation of non-thermal MRgFUS ablation of brain tumors to large animal models and ultimately to the clinic. We will achieve this in two specific aims; 1) Develop innovative MRI-based approaches for direct real-time monitoring of tissue of interest during non- thermal ablation, and evaluate them based on real-time applicability and sensitivity to acutely detect lesion formation in a rat brain tumor model; 2) Adapt an MRI to histopathology registration pipeline and use it to gain understanding about lesion progression and to develop imaging biomarkers for acute prediction of long-term tissue non-viability. The research proposed in this application is innovative, in the applicant’s opinion, because it will develop sensitive approaches for real-time monitoring of the lesion progression of FUS non-thermal ablation, and will use a MRI-to-histology registration pipeline to develop understanding about lesion progression and develop imaging biomarkers to predict tissue nonviability. Ultimately, this novel non-invasive treatment modality has the potential to provide the tens of thousands of patients diagnosed with brain tumors each year with a safe an efficient treatment option.