The authors describe a method to enhance the photostability of fluorescent proteins, which is crucial for applications requiring repeated imaging of single cells. They developed a high-throughput screening assay using a solar simulator to photobleach bacterial colonies and select for more stable variants. This method was applied to two fluorescent proteins, mOrange and TagRFP, resulting in highly stable variants, mOrange2 and TagRFP-T, respectively. These variants maintain most of the desirable properties of their original forms while being significantly more photostable. The study also highlights the importance of specific residues near the chromophore in determining photostability and discusses the potential for reversible photoswitching in these new variants. The authors conclude that their screening method can be applied to other fluorescent proteins to improve their photostability, making them more suitable for advanced imaging applications.The authors describe a method to enhance the photostability of fluorescent proteins, which is crucial for applications requiring repeated imaging of single cells. They developed a high-throughput screening assay using a solar simulator to photobleach bacterial colonies and select for more stable variants. This method was applied to two fluorescent proteins, mOrange and TagRFP, resulting in highly stable variants, mOrange2 and TagRFP-T, respectively. These variants maintain most of the desirable properties of their original forms while being significantly more photostable. The study also highlights the importance of specific residues near the chromophore in determining photostability and discusses the potential for reversible photoswitching in these new variants. The authors conclude that their screening method can be applied to other fluorescent proteins to improve their photostability, making them more suitable for advanced imaging applications.