In biomedical imaging, nanoparticles combined with radionuclides that generate Cerenkov luminescence are used in diagnostic imaging, photon-induced therapies, and as activatable probes. In these applications, the nanoparticle is often viewed as a carrier inert to ionizing radiation from the radionuclide. However, certain phenomena such as enhanced nanoparticle luminescence and generation of reactive oxygen species cannot be explained by only Cerenkov luminescence interactions with nanoparticles. Herein, we report methods to examine the mechanisms of nanoparticle excitation by radionuclides, including interactions with Cerenkov luminescence, β particles, and γ radiation. We demonstrate that β scintillation contributes appreciably to excitation and reactivity in certain nanoparticle systems and that excitation of nanoparticles composed of large atomic number atoms by radionuclides generates X-rays, enabling multiplexed imaging through single photon emission computed tomography. These findings demonstrate practical optical imaging and therapy using radionuclides with emission energies below the Cerenkov threshold, thereby expanding the list of applicable radionuclides.
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