Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization m...Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization microscopy(SMLM)with self-blinking dyes.However,existing self-blinking dyes often face limitations,such as nonspecific blinking and low photostability,which can bring background noise and yield erroneous localization signals,hindering their effectiveness for nanoscale visualization.Here,we present a method for long-term SMLM imaging of cellular pseudopodia dynamics using a blinkogenic probe that exhibits self-blinking activation upon molecular recognition.This approach enabled the precise tracking of various pseudopodia structures,including filopodia,lamellipodia,and(tunneling nanotubes)-nanoscale(TNTs),in living cells.We monitored the growth and fusion of filopodia,as well as the extension and shrinkage of lamellipodia,in real-time.Additionally,we identified two distinct fusion modes between filopodia and lamellipodia and captured the formation of TNTs and their interactions with filopodia,demonstrating the probe's utility in visualizing real-time pseudopodia dynamics at nanoscale.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.DICPI202227,DICPI202436)。
文摘Monitoring the dynamics of cellular pseudopodia at nanoscale has become essential for understanding their diverse and complex functions in living cells.This is made possible by combining single-molecule localization microscopy(SMLM)with self-blinking dyes.However,existing self-blinking dyes often face limitations,such as nonspecific blinking and low photostability,which can bring background noise and yield erroneous localization signals,hindering their effectiveness for nanoscale visualization.Here,we present a method for long-term SMLM imaging of cellular pseudopodia dynamics using a blinkogenic probe that exhibits self-blinking activation upon molecular recognition.This approach enabled the precise tracking of various pseudopodia structures,including filopodia,lamellipodia,and(tunneling nanotubes)-nanoscale(TNTs),in living cells.We monitored the growth and fusion of filopodia,as well as the extension and shrinkage of lamellipodia,in real-time.Additionally,we identified two distinct fusion modes between filopodia and lamellipodia and captured the formation of TNTs and their interactions with filopodia,demonstrating the probe's utility in visualizing real-time pseudopodia dynamics at nanoscale.
