The advent of RNA vaccines has dramatically changed the immunological landscape. RNA vaccines have had remarkable success in the COVID pandemic and shown great promise in personalized cancer immunotherapy leveraging the patient’s own immune system. Given the importance and potential of such vaccines, it is imperative to understand the fundamental principles underlying vaccine formulation for effective and precision medicine. Our real-time fluorescence imaging provides unprecedented insight into the assembly process of lipid-based RNA nanoparticles, which is the basis of our mRNA vaccine formulation currently being used in patient trials at UF. We show that a diffusion-limited-assembly process underlies the formation of heterogeneous conglomerates spanning ~1-100 micrometers in size. Further, leveraging physical insights driven by high-resolution imaging and advanced image analysis, we have shown modulating the assembly process can alter the efficiency of antigen expression in dendritic cells. The results of this experiment will show the power of direct imaging of the microscopic assembly process which may open up new opportunities for vaccine formation optimization.