Persistent　luminescent　nanoparticles　(PLNPs)　have　emerged　as　important　nanomaterials　for　biological　imaging　as　a　result　of　complete　avoidance　of　tissue　auto-fluorescence.　However,　the　imaging　sensitivity　and　long-term　in　vivo　imaging　are　still　limited　due　to　the　persistent　luminescence　that　is　rapidly　decayed　in　vivo　after　an　ex　vivo　excitation.　To　address　this　limitation,　in　vivo　activation　of　PLNPs　is　highly　desired.　Herein,　we　present　a　new　strategy　for　the　activation　of　PLNPs　(SrAl2O4:Eu2+)　by　using　soft　X-ray　excitation.　Importantly,　as　the　soft　X-ray　light　source　possesses　the　advantage　of　deep　tissue　penetration,　the　PLNPs　can　be　reactivated　in　vivo　through　living　tissue　using　soft　X-ray　excitation.　Furthermore,　X-ray/persistent　luminescence　dual-modal　imaging　can　be　achieved　to　empower　this　strategy　with　high　sensitivity.　Our　results　suggest　that　this　new　strategy　of　in　vivo　energy　charging　in　PLNPs　would　bring　new　insights　for　deep　tissue　and　long-term　bioimaging　in　living　animals,　and　provide　new　perspectives　for　persistent　luminescence　bioimaging　and　therapeutic　applications.　©　The　Royal　Society　of　Chemistry　2017.