A photoactivatable push-pull fluorophore for single-molecule imaging in live cells

Area of Science:

• Organic chemistry
• Biophysics
• Microscopy

Background:

• Developing novel fluorophores is crucial for advanced imaging techniques.
• Photoactivatable fluorophores offer temporal control over signal generation.
• Existing bright photoactivatable fluorophores are limited for certain super-resolution applications.

Purpose of the Study:

• To engineer a novel red-emitting dicyanomethylenedihydrofuran fluorophore.
• To achieve photoactivation of a dark fluorogen using low-intensity violet light.
• To demonstrate its utility for single-molecule imaging in living cells.

Main Methods:

• Reengineering a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore.
• Utilizing a short burst of low-intensity violet light for photoactivation.
• Observing the conversion of an azide to an amine upon photoactivation.
• Assessing brightness and photostability for single-molecule imaging.

Main Results:

• The reengineered fluorophore is initially dark and becomes red-emitting upon photoactivation.
• Photoactivation involves the conversion of an azide to an amine, causing a spectral shift.
• The activated fluorophore exhibits high brightness and photostability.
• Successful single-molecule level imaging was achieved in living cells.

Conclusions:

• A new class of bright, photoactivatable red-emitting fluorophores has been demonstrated.
• This fluorophore enables precise control over emission through photoactivation.
• The developed fluorophore is suitable for advanced super-resolution imaging requiring single-molecule emission control.