Super-beacons: Open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution...

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Localization-based super-resolution microscopy requires molecules to switch between fluorescent and non-fluorescent states.
• Current methods often use high-intensity light, causing photodamage and limiting imaging hardware.
• Existing probes may require toxic buffers for effective photoswitching.

Purpose of the Study:

• To introduce single-molecule self-quenching as a novel mechanism for spontaneous photoswitching in super-resolution microscopy.
• To develop and characterize a new class of DNA-based super-resolution probes, termed super-beacons.
• To demonstrate the application of super-beacons for live-cell imaging without harsh illumination or chemical additives.

Main Methods:

• Development of DNA-based super-resolution probes (super-beacons) engineered for tunable photoswitching kinetics.
• Investigation of photoswitching mechanisms, including structural, thermal, and chemical modulation.
• Application of super-beacons in live-cell super-resolution microscopy to image interferon inducible transmembrane proteins (IFITMs).

Main Results:

• Demonstrated spontaneous photoswitching in super-beacons via single-molecule self-quenching.
• Showcased tunable photoswitching kinetics of super-beacons through structural, thermal, and chemical modifications.
• Achieved sub-100 nm resolution imaging of IFITMs in live cells using super-beacons without high illumination or toxic buffers.

Conclusions:

• Single-molecule self-quenching offers a viable alternative for generating photoswitching in super-resolution microscopy.
• Super-beacons represent a versatile and open-source platform for advanced super-resolution imaging.
• This approach significantly reduces photodamage and toxicity, paving the way for improved live-cell super-resolution studies.