Ultrabright photoactivatable fluorophores created by reductive caging

Area of Science:

• Biophysics
• Optical Microscopy
• Chemical Biology

Background:

• Super-resolution microscopy relies on precise localization of photoactivatable fluorophores.
• Image resolution is directly correlated with the number of photons detected per fluorophore localization.
• Existing photoactivatable probes may have limitations in photon yield, impacting achievable resolution.

Purpose of the Study:

• To develop a novel strategy for creating photoactivatable probes with significantly enhanced photon yields.
• To improve the resolution limits of super-resolution imaging techniques.
• To demonstrate the broad applicability of the developed fluorophore caging strategy.

Main Methods:

• A new fluorophore caging strategy was designed and implemented to create photoactivatable probes.
• The photon yield per localization of the developed probes was quantified.
• The developed probes were utilized for imaging fixed biological samples using super-resolution microscopy techniques.
• The achievable resolution with the new probes was assessed.

Main Results:

• The developed caging strategy resulted in photoactivatable probes with high photon yields, ranging from 10^4 to 10^6 photons per localization.
• Super-resolution imaging of fixed samples using these probes achieved resolutions of several nanometers.
• The strategy proved effective across a range of fluorophores emitting in the visible spectrum.

Conclusions:

• The developed fluorophore caging strategy is a powerful approach to enhance photoactivatable probes for super-resolution imaging.
• This method significantly improves photon yield, leading to nanometer-scale resolution in biological imaging.
• The versatility of the strategy across various fluorophores makes it broadly applicable for advancing optical microscopy.