Abstract
Abstract Photocytotoxicity represents a significant limitation in the application of dye‐assisted fluorescence imaging (FI), often resulting in undesirable cellular damage or even cell death, thereby restricting their practical utility. The prevalence of Rhodamine B (RhB) in FI underscores the importance of elucidating its photocytotoxicity effects to minimize photodamage. This study identifies the primary cause of photocytotoxicity stems from the generation of cytotoxic singlet oxygen in RhB, utilizing femtosecond transient absorption spectroscopy coupled with quantum chemical calculations. The Laser power‐dependent cellular viability reveals a threshold at about 50 mW cm−2, surpassing which produces pronounced photocytotoxicity in vitro and in vivo. Notably, this threshold significantly falls below the safety limits (<200 mW cm−2) for laser use in health care, implying a huge risk of photodamage. This study provides valuable insights into the photocytotoxicity and offers essential guidelines for developing safer imaging protocols.