A Photoactivatable Plasma Membrane Probe Based on a Self-Triggered Photooxidation Cascade for Live Cell...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Super-resolution microscopy requires precise control over fluorophore activation and deactivation.
• Photoactivatable fluorophores are crucial for achieving this control by switching between ON and OFF states upon light exposure.

Purpose of the Study:

• To introduce a novel self-triggered photooxidation cascade (STPC) for enhanced single-molecule localization microscopy.
• To enable super-resolution imaging of live cellular structures with improved spatio-temporal control.

Main Methods:

• Utilized a plasma membrane-targeted leuco-rhodamine (LRhod-PM) probe, a non-fluorescent precursor.
• Irradiated with visible light to initiate photooxidation, generating singlet oxygen.
• Leveraged the generated singlet oxygen to convert OFF-state LRhod-PM to its fluorescent ON-state Rhod-PM.

Main Results:

• Demonstrated that STPC is kinetically favored by high local probe concentration within membrane bilayers.
• Observed rapid propagation of the photooxidation cascade in membrane environments.
• Showcased that close proximity of dyes enhances photobleaching, facilitating a single-molecule blinking regime.

Conclusions:

• The STPC method achieves a single-molecule blinking regime through concomitant activation and bleaching.
• Enabled super-resolution imaging of live cellular membranes, including fine structures like filopodia and tunneling nanotubes.
• Provides a new strategy for spatio-temporal control in super-resolution microscopy of biological systems.