FLEXTAG: a small and self-renewable protein labeling system for anti-fading multi-color super-resolution imaging

Area of Science:

• Cell Biology
• Microscopy
• Biochemistry

Background:

• Super-resolution fluorescence imaging offers nanoscale visualization but is limited by protein tagging systems.
• Existing tags suffer from photobleaching, artifacts, poor labeling, and limited multiplexing.

Purpose of the Study:

• To develop a comprehensive protein labeling system to overcome limitations in super-resolution imaging.
• To enable optimized multi-color super-resolution imaging with enhanced stability and efficiency.

Main Methods:

• Introduced FLEXTAG, a system with three orthogonal, ultrasmall, self-renewable protein tags.
• Utilized continuous fluorophore exchange for extended imaging and minimal photobleaching.
• Developed protection-based fixation and chemical blocking for improved labeling and signal-to-noise ratio.

Main Results:

• FLEXTAG overcomes photobleaching, tag artifacts, and labeling inefficiencies.
• Achieved optimized multi-color super-resolution imaging compatible with SIM, STED, STORM, and PAINT.
• Demonstrated long-term tracking of dynamic cellular behaviors and nanoscale organization.

Conclusions:

• FLEXTAG significantly advances super-resolution imaging capabilities for live and fixed cells.
• The developed fixation and blocking strategies offer broad applicability to other tagging systems.
• Enables enhanced basic research and translational applications in cell biology.