Expansion microscopy (ExM) enhances spatial resolution by using a swellable polymer that expands the sample volume by a factor of ≈4 in one dimension and a factor of ≈64 in volume. Combining ExM with stimulated emission depletion (STED) microscopy, referred to as ExSTED, increases the resolution to up to 10 nm. 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 GW cm−2. We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle.
Bibliographical noteFunding Information:
We thank the Hyunwoo Rhee Lab (Seoul National University) for providing biotin ligase and APEX2 plasmids and the Hak Joong Kim Lab (Korea University) for providing the Avi plasmids. This work is supported by IBS-R023-D1. S.-H.S., and D.Y.K also appreciates the support of the Samsung Science and Technology Foundation (SSTF-BA1501-08) and Korea University (K1707381). All STED microscopy measurements were performed at the Korea Basic Science Institute.
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- expansion microscopy
- stimulated emission depletion microscopy
- super-resolution microscopy
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
- Organic Chemistry