This study describes the development of highly photostable variants of two fluorescent proteins, mOrange and TagRFP, through directed evolution and screening methods. The researchers aimed to improve the photostability of these proteins, which is crucial for experiments requiring high image resolution and long exposure times. They developed a screening assay that allows for the selection of fluorescent proteins with enhanced photostability while maintaining acceptable fluorescence levels. The study focused on two fluorescent proteins: mOrange, a monomeric orange fluorescent protein, and TagRFP, a monomeric red fluorescent protein. Through directed evolution, the researchers identified variants of these proteins with significantly improved photostability. For mOrange, the variant mOrange2 was developed, which is 25-fold more photostable than the original mOrange. For TagRFP, the variant TagRFP-T was developed, which is 9-fold more photostable than the original TagRFP. Both variants maintain most of the beneficial properties of their original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs. The researchers also evaluated the photostability of these variants under various conditions, including arc lamp illumination and laser scanning confocal microscopy. The results showed that mOrange2 is over 25-fold more photostable than mOrange and nearly twice as photostable as mKO, the previously most photostable orange monomer. TagRFP-T is also highly photostable and suitable for co-imaging with green fluorescent proteins. The study also addressed concerns about reversible photoswitching in the new fluorescent proteins. While some fluorescent proteins exhibit reversible photoswitching, the new variants showed less severe photoswitching behavior. The researchers also evaluated the performance of the new variants in various fusion constructs and found that they perform well in many applications. Overall, the study demonstrates that photostability can be improved through directed evolution and screening methods, leading to the development of highly photostable fluorescent proteins that are useful for a wide range of applications in biological research.This study describes the development of highly photostable variants of two fluorescent proteins, mOrange and TagRFP, through directed evolution and screening methods. The researchers aimed to improve the photostability of these proteins, which is crucial for experiments requiring high image resolution and long exposure times. They developed a screening assay that allows for the selection of fluorescent proteins with enhanced photostability while maintaining acceptable fluorescence levels. The study focused on two fluorescent proteins: mOrange, a monomeric orange fluorescent protein, and TagRFP, a monomeric red fluorescent protein. Through directed evolution, the researchers identified variants of these proteins with significantly improved photostability. For mOrange, the variant mOrange2 was developed, which is 25-fold more photostable than the original mOrange. For TagRFP, the variant TagRFP-T was developed, which is 9-fold more photostable than the original TagRFP. Both variants maintain most of the beneficial properties of their original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs. The researchers also evaluated the photostability of these variants under various conditions, including arc lamp illumination and laser scanning confocal microscopy. The results showed that mOrange2 is over 25-fold more photostable than mOrange and nearly twice as photostable as mKO, the previously most photostable orange monomer. TagRFP-T is also highly photostable and suitable for co-imaging with green fluorescent proteins. The study also addressed concerns about reversible photoswitching in the new fluorescent proteins. While some fluorescent proteins exhibit reversible photoswitching, the new variants showed less severe photoswitching behavior. The researchers also evaluated the performance of the new variants in various fusion constructs and found that they perform well in many applications. Overall, the study demonstrates that photostability can be improved through directed evolution and screening methods, leading to the development of highly photostable fluorescent proteins that are useful for a wide range of applications in biological research.