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 ≈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.

Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

“…A variant of this method allows signal amplification through iterative treatment of the sample with biotin‐ and dye‐conjugated secondary antibodies and dye‐conjugated streptavidin. While this method gives a fluorophore density high enough to reach the maximum STED resolution in an expanded sample, it also introduces a localisation error with each amplification cycle, due to the resulting distance between the protein of interest and the fluorophore [20] . An alternative to expressing the protein of interest conjugated with a fluorescent protein is to attach the AviTag peptide sequence, consisting of only 16 amino acids.…”

“…The small peptide is less likely to interfere with the proteins motility and to block binding domains than the bulky structures of a fluorescent protein. This previously demonstrated method [20] then uses the BirA biotin ligase to biotinylate the peptide. The sample can then be treated with the same iterative treatment as outlined above.…”

Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy

“…Combining different SRM methods, or SRM with other imaging or experimental approaches, has proven effective at harnessing the advantages of each. For example, STED microscopy has been combined with expansion microscopy (ExSTED; Gao et al, 2018;Kim et al, 2019), allowing further resolution gains to under 10 nm. This approach could be particularly relevant in the extremely crowded space of synaptic compartments, particularly if using probes that are smaller than antibodies.…”

Stimulated emission depletion microscopy for biological imaging in four dimensions: A review

“…ExSTED imaging of microtubules in BS‐C‐1 cell line achieves 18 nm resolution (R. Li, Chen, Lin, Wang, & Sun, 2018). Following this trend, extremely bright expansion nanoscopy was designed by using biotin–avidin signal amplification to increase the labeling density and to reduce photobleaching in ExSTED, serving as evolutive ExSTED to possess 10 nm resolution (Kim, Kim, Lee, & Shim, 2019).…”