To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) Solution into the yolk of embryo and Visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT. were irradiated with 5 Gy X-rays which is a non-lethal close for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase Lip to 24 h after irradiation and completely disappeared Lip to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.