Amplified Expansion Stimulated Emission Depletion Microscopy

Abstract

Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of approximate to 4 in one dimension and a factor of approximate to 64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10nm. However, photobleaching is a critical issue in ExSTED because the sample expansion lowers the fluorophore density whereas high-resolution STED requires high depletion intensity. To overcome these issues, we developed extremely bright expansion nanoscopy by using biotin-avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GWcm(-2). We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.