Dual-Color Expansion Microscopy of Membrane Proteins Using Bioorthogonal Labeling.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Fluorescence microscopy resolution is often limited by label size and linkage errors, not the microscope.
• Antibody-based probes introduce spatial uncertainty in fluorescent protein labeling.
• Site-specific ncAA incorporation with bioorthogonal click chemistry offers improved labeling precision.

Purpose of the Study:

• To develop and validate a method combining ncAA labeling and expansion microscopy (ExM) for dual-color super-resolution imaging.
• To enhance labeling precision beyond current antibody-based methods.
• To achieve nanoscale visualization of specific protein subunits.

Main Methods:

• Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins.
• Utilizing bioorthogonal click chemistry for fluorescent labeling.
• Applying expansion microscopy (ExM) to increase sample size and improve resolution.
• Performing super-resolution STED imaging for validation.

Main Results:

• Optimized ncAA labeling procedures and fluorophore selection were achieved.
• Successfully visualized and resolved the nanoscale distribution of Na,K-ATPase α1 and β1 subunits in expanded HEK 293T cells.
• Validated the approach using STED imaging on unexpanded, ncAA-labeled cells.

Conclusions:

• The combination of ncAA labeling and ExM provides a robust framework for multiplexed, high-resolution imaging.
• This method enables biological imaging at the nanometer scale, overcoming previous resolution limitations.
• The developed technique significantly improves labeling precision for super-resolution microscopy.