Microphysiological tumor models (?PTM) are increasingly used for preclinical research due to their ability to closely simulate, in vitro, the physiology of solid tumors. With the advent of microfluidics technology, new methods have been introduced to grow tissues in 3D inside perfused chambers and precisely control biological factors, such as cells, nutrients and oxygen, at a spatial and temporal level. These models can incorporate 3D extracellular matrices (ECM) and perfusable neovasculature, both key components of solid tumors. Being optically transparent, they permit excellent visualization of live cells through advanced optical microscopy techniques. Radioluminescence microscopy (RLM) is a method that was developed to image clinical radiotracers in live cells with high spatial resolution. However, this method in its current form cannot be used to adequately image 3D cell cultures due to the loss of spatial resolution and lack of tomographic capabilities for imaging thick samples. The goal of this project is to develop a novel layered scintillator design for limited-angle tomographic imaging of 3D cell cultures and other in vitro tissues such as organoids and tumor-chips. The dual-layer scintillator will provide angular information that can be used for 3D reconstruction of radiotracer distribution in these thick samples. Thus, such a technological advance has the potential for widespread use in research and medicine using the arsenal of existing diagnostic and therapeutic radioisotopes. It could be used to bridge the gap between these emergent tumor models and clinical trials, which use PET biomarkers as disease endpoints. In addition, the technology could be used to characterize how properties specific to the 3D microenvironment surrounding microtumors could affect the uptake and retention of radiotracers. Higher spatial resolution will allow cells to be probed in situ, in dense tissue sections. These new capabilities will be critical to help researchers develop patient-derived tumor models that recapitulate the most salient features of solid tumors and can be imaged using clinically relevant PET tracers. The objective of this Phase I project is to demonstrate the feasibility of successfully fabricating thin layers of a highly dense transparent scintillator, separated by a layer of non-scintillating transparent material. This novel design enables visualization of two scintillation spots so that the angle of incidence can be estimated to provide limited-angle tomographic projections. This innovative design will provide the spatial resolution required for visualization of radiotracer uptake in 3D cell cultures, microtumors, and other thick specimens.