Fluorogen-activating proteins(FAPs)selectively bind to specific fluorophores,inducing fluorescence activation through the inhibition of torsion of fluorophores.This binding-activation mechanism provides a highly speci...Fluorogen-activating proteins(FAPs)selectively bind to specific fluorophores,inducing fluorescence activation through the inhibition of torsion of fluorophores.This binding-activation mechanism provides a highly specific and efficient fluorescence system that minimizes background signals,significantly enhancing the signal-to-noise ratio(SNR)and making it a powerful tool in live-cell imaging.The principle of binding-activation fluorescence is fundamental to point accumulation for imaging in nanoscale topography(PAINT)super-resolution imaging.However,the high binding affinity between traditional FAPfluorophore pairs limits their application in PAINT,thus hindering the rapid and dynamic imaging necessary for high-resolution cellular studies.In this work,we designed malachite green(MG)derivatives with bulky N-substituents to modulate the binding affinity of the MG-d L5^(**)fluorophore-FAP pair.This modification introduces steric hindrance in MG-dL5^(**)system,resulting in reduced binding affinity and practicability for fast,high-resolution PAINT imaging.Among the synthesized derivatives,MG-Pen showed optimal properties,enabling rapid and high-resolution PAINT imaging of dL5^(**)in living cells.This study highlights the potential of MG derivatives optimization in overcoming the limitations of fluorophore-FAP pairs for super-resolution imaging and provides a new approach for enhancing the performance of PAINT in living cell applications.展开更多
基金supported by the National Natural Science Foundation of China(Nos.22225806,22078314,22278394,22378385)Dalian Institute of Chemical Physics(Nos.DICPI202142,DICPI202436)。
文摘Fluorogen-activating proteins(FAPs)selectively bind to specific fluorophores,inducing fluorescence activation through the inhibition of torsion of fluorophores.This binding-activation mechanism provides a highly specific and efficient fluorescence system that minimizes background signals,significantly enhancing the signal-to-noise ratio(SNR)and making it a powerful tool in live-cell imaging.The principle of binding-activation fluorescence is fundamental to point accumulation for imaging in nanoscale topography(PAINT)super-resolution imaging.However,the high binding affinity between traditional FAPfluorophore pairs limits their application in PAINT,thus hindering the rapid and dynamic imaging necessary for high-resolution cellular studies.In this work,we designed malachite green(MG)derivatives with bulky N-substituents to modulate the binding affinity of the MG-d L5^(**)fluorophore-FAP pair.This modification introduces steric hindrance in MG-dL5^(**)system,resulting in reduced binding affinity and practicability for fast,high-resolution PAINT imaging.Among the synthesized derivatives,MG-Pen showed optimal properties,enabling rapid and high-resolution PAINT imaging of dL5^(**)in living cells.This study highlights the potential of MG derivatives optimization in overcoming the limitations of fluorophore-FAP pairs for super-resolution imaging and provides a new approach for enhancing the performance of PAINT in living cell applications.
