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

• Published in: Analyst (London) 2021-12, Vol.147 (1), p.139-146
• Main Authors: Kuang, Weibing, Xin, Bo, Huang, Zhen-Li, Shi, Bing
• Format: Article
• Language: English
• Subjects: Antibodies; Biological properties; Biotin; Buffers; Chemical compounds; Fluorescence; Fluorescent Dyes; Hydrogels; Labeling; Labelling; Localization; Medical research; Microscopy; Optical microscopy; Organelles; Polyacrylamide; Proteins; Resolution; Single Molecule Imaging; Streptavidin
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/d1an01680f

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 in 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 an ionic photoswitching buffer, fluorescence photobleaching due to a free-radical reaction and reduced labelling efficiency from protease digestion. Re-embedding polyacrylamide gel or using an improved photoswitching buffer with a low ionic strength is 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 introducing 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 were able to label protein epitopes with biotin, which was stable during the expansion process, and thus avoid the troublesome problem in preserving protein epitopes or antibodies. We demonstrate that combining Ex-SMLM with the new post-expansion linkage scheme enables new possibilities in resolving the detailed arrangement of Nup133 proteins in the nuclear pore complex, which helps researchers to observe a clearer structure. This study provides new opportunities for studying the ultrastructural details of subcellular organelles or even biomacromolecules, using the conventional SMLM system. We introduce a simplified labeling strategy based on biotin-streptavidin interactions for expansion single-molecule localization microscopy (Ex-SMLM) is proposed, which can effectively preserve fluorophores and improve the resolution of SMLM 2.6 times.