Controlled Grafting Expansion Microscopy.

Area of Science:

• Biotechnology
• Microscopy
• Molecular Biology

Background:

• Expansion microscopy (ExM) offers super-resolution imaging by physically expanding hydrogel-embedded biological samples.
• A key challenge in ExM is the loss of fluorescent labels during hydrogel formation and digestion, leading to weak signals.
• Existing methods struggle to retain sufficient label integrity for high-resolution imaging.

Purpose of the Study:

• To develop a novel agent that combines targeting, fluorescent labeling, and hydrogel linkage in a single molecule for improved ExM signal retention.
• To address the issue of label loss caused by insufficient surface grafting of fluorophores into the hydrogel.
• To enhance the resolution of cellular structures in ExM by improving fluorescence signal.

Main Methods:

• Development of a novel small molecule agent integrating targeting, fluorescent labeling, and gel linkage functionalities.
• Modification of ExM protocols to increase the surface grafting of target-bound monomers into the hydrogel.
• Comparative analysis of fluorescence signal retention and imaging resolution before and after applying the new agent.

Main Results:

• The novel agent significantly improves fluorescence signal retention compared to previous methods.
• Increased surface grafting of target-bound monomers effectively reduces label loss during ExM processing.
• The improved signal allows for the high-resolution visualization of nuclear pores as distinct ring-like structures, comparable to STED microscopy.
• Mechanistic insights into dye retention within the ExM hydrogel were elucidated.

Conclusions:

• The developed small molecule agent is effective in overcoming signal loss in expansion microscopy.
• Enhanced hydrogel integration of fluorescent labels leads to superior imaging performance in ExM.
• This advancement enables clearer, high-resolution imaging of subcellular structures, advancing super-resolution microscopy techniques.