Super-resolution labelling with Action-PAINT

Area of Science:

• Molecular Biology
• Microscopy
• Nanotechnology

Background:

• Super-resolution microscopy enables visualization of nanoscale molecular features (~5 nm).
• Current methods lack user-prescribed, context-dependent manipulation of single molecules.
• Need for precise, real-time molecular labeling and manipulation in biological systems.

Purpose of the Study:

• To introduce and validate the Action-PAINT strategy for super-resolution labeling.
• To enable user-prescribed, context-dependent manipulation of single molecules.
• To achieve real-time, targeted nanoscale labeling and molecular manipulation.

Main Methods:

• Developed the 'Action-PAINT' (point accumulation for imaging in nanoscale topography) strategy.
• Utilized DNA-PAINT for real-time monitoring and localization of DNA binding events.
• Employed 3-cyanovinylcarbazole nucleoside for photo-inducible crosslinking of DNA labels.
• Created software for real-time super-resolution imaging and crosslinking control.

Main Results:

• Demonstrated efficient photo-crosslinking of single molecular targets.
• Achieved targeted multipoint labeling on DNA nanostructures with 30 nm selectivity.
• Successfully performed in situ labeling on fixed microtubule samples with 40 nm target size and controlled subdiffraction spacing.

Conclusions:

• Action-PAINT provides a novel method for super-resolution labeling and manipulation of single molecules.
• The technique allows for precise, real-time control over molecular labeling in complex biological samples.
• This approach significantly advances nanoscale imaging and molecular manipulation capabilities.