Amplified Expansion Stimulated Emission Depletion Microscopy

Area of Science:

• Biotechnology
• Microscopy
• Cell Biology

Background:

• Expansion microscopy (ExM) improves spatial resolution by physically expanding samples.
• Stimulated emission depletion (STED) microscopy achieves nanoscale resolution but is prone to photobleaching.
• Combining ExM and STED (ExSTED) enhances resolution but exacerbates photobleaching due to reduced fluorophore density and high laser power.

Purpose of the Study:

• To develop a method to overcome photobleaching in ExSTED imaging.
• To increase signal intensity in expanded samples for super-resolution microscopy.
• To enable high-resolution STED imaging with commercially available equipment.

Main Methods:

• Implemented biotin-avidin signal amplification to increase fluorophore labeling density.
• Utilized biotinylated antibodies and genetic incorporation for targeted biotin localization.
• Applied STED microscopy with high depletion intensities on expanded samples.

Main Results:

• Achieved up to a sevenfold increase in fluorescence signal intensity in expanded samples.
• Enabled STED imaging at high depletion intensities (GW cm⁻²) without significant photobleaching.
• Demonstrated successful super-resolution localization of specific molecules and organelles.

Conclusions:

• Biotin-avidin signal amplification effectively enhances brightness in expansion nanoscopy.
• This approach overcomes photobleaching limitations in ExSTED, facilitating routine super-resolution imaging.
• The developed method expands the applicability of high-resolution microscopy techniques.