Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy

Area of Science:

• Biotechnology
• Microscopy
• Cell Biology

Background:

• Expansion microscopy (ExM) enables super-resolution imaging on standard microscopes.
• Efficient visualization of actin filaments using ExM remains challenging.
• Previous attempts to improve actin imaging with multifunctional molecules yielded moderate success.

Purpose of the Study:

• To develop optimized methods for phalloidin conjugate grafting for enhanced actin imaging in ExM.
• To introduce and evaluate trifunctional linkers (TRITON) for improved actin visualization.
• To demonstrate the efficacy of TRITON linkers in various ExM protocols and sample types.

Main Methods:

• Optimized phalloidin conjugate grafting strategies.
• Development and application of trifunctional linkers (TRITON).
• Imaging of actin structures in cultured neurons and brain slices using ExM and Airyscan confocal microscopy.

Main Results:

• The optimized grafting strategy improved anchoring and signal retention by approximately 10 times.
• TRITON linkers demonstrated high efficiency for actin imaging in both cellular and tissue samples.
• 10X ExM with TRITON-labeled actin allowed visualization of actin ring periodicity in neurons and brain slices.

Conclusions:

• TRITON linkers provide an efficient method for actin visualization in ExM.
• This approach is particularly beneficial when target-bound monomer concentration is low.
• Optimized ExM with TRITON enhances the study of actin-rich structures in biological samples.