A labeling strategy with effective preservation of fluorophores for expansion single-molecule localization microscopy (Ex-SMLM).

Expansion Microscopy (ExM) significantly improves the resolution of conventional diffraction-limited optical microscopy by using physically expanding biological samples. Combining ExM with single molecule localization microscopy (SMLM) could further enhance the resolving power of SMLM, which is typically on the order of 20-30 nm. However, to make this combination successful, we need to solve three key issues related to sample preparation, including mainly hydrogel shrinking in ionic photoswitching buffer, fluorescence photobleaching due to free-radical reaction and reduced labelling efficiency from protease digestion. Using polyacrylamide gel re-embedding or improved photoswitching buffer with low ionic strength are able to minimize or even solve the hydrogel shrinking problem, while the development of post-expansion labelling approaches avoids fluorescence bleaching. However, the preservation of protein epitopes (which determines the labelling efficiency) remains to be challenging. In this paper, we propose to tackle this challenge by inserting the highly selective and stable biotin-streptavidin interaction into the post-expansion labelling strategy. After upgrading the popular immunolabelling linkage scheme from Epitope-Primary antibody-Secondary antibody-Fluorophores to Epitope-Primary antibody-Secondary antibody-Biotin-Streptavidin-Fluorophores, we are able to label protein epitopes with Biotin, which is stable during the expansion process, and thus avoid the troublesome problem in preserving protein epitopes or antibody. We demonstrate that combining Ex-SMLM with the new post-expansion linkage scheme enables new possibility in resolving the detailed arrangement of the Nup133 proteins in nuclear pore complex, which helps researchers observe a clearer structure. This study provides new chances for studying the ultrastructural details of subcellular organelles or even biomacromolecules, using conventional SMLM system.