Despite the large interest on nuclear/optical multimodality imaging, the effect of radiation on the fluorescence of fluorophores remains unexplored. Herein we determined the radiosensitivity of two near infrared fluorescent compounds, IRDye 800CW (800CW) and a dual modality imaging tetrapeptide containing DOTA as chelator and Dylight 800 as fluorophore, exposed to increasing activities of 111In, 68Ga, or 213Bi (γ, β, and α emitter, respectively). An activity and type of radiation-dependent radiation-induced loss of fluorescence, radiobleaching, of 800CW was observed upon incubation with escalating activities of 111In, 68Ga, or 213Bi. 68Ga showed the largest radiolytic effect, followed by 111In and 213Bi. The addition of oxygen radical scavengers including ethanol, gentisic acid, and ascorbic acid (AA), provided a concentration dependent radioprotective effect. These results supported the hypothesis of a free radical-mediated
radiobleaching mechanism. AA provided the most robust radioprotection over a wide range of concentrations and preserved
fluorescence at much higher radioactivity levels. Overall, both fluorescent compounds displayed similar sensitivity, except for 213Bi-irradiated solutions, where the dual modality construct exhibited enhanced radiolysis, presumably due to direct radiation damage from α particles. Concurrently, AA was not able to preserve fluorescence of the dual-modality molecule labeled with 213Bi. Herein we report on the radiobleaching of fluorophores, and describe conditions to provide robust radioprotection under practical (pre)clinical conditions. Our recommendations have strong repercussions for the preparation of dual-modality radiopharmaceuticals.