We describe the development of click-expansion microscopy (Click-ExM), which integrates click-labeling into ExM to enable a"one-stop-shop" method for nanoscale imaging of various types of biomolecules.Using 18 clickable labels for click-ExM imaging of DNA, RNA, proteins, lipids, glycans and small molecules, we demonstrate its universality, compatibility with signal-ampli cation techniques, and broad applications in cellular and tissue imaging. The click-labeling and ExM steps could be nished within two days. This step-by-step protocol is related to the publication "Click-ExM enables expansion microscopy for all biomolecules" in Nature Methods.

Adipose tissue is an active endocrine organ that can secrete a wide number of factors to regulate adipogenesis via paracrine signals. In addition to soluble proteins in adipose tissue, microRNAs (miRNAs) enriched in extracellular vesicles (EVs), such as exosomes or microvesicles, could modulate intercellular communications. In this study, we demonstrated that exosome-like vesicles derived from adipose tissue (Exo-AT) were internalized by adipose tissue-derived stem cells (ADSCs), and that these, in turn, induced adipogenesis. High-throughput sequencing showed that 45 miRNAs were enriched in Exo-AT, and 31.11% of them were associated with adipogenesis, compared with ADSC-derived exosome-like vesicles (Exo-ADSC). miR-450a-5p, one of the most abundant miRNAs in Exo-AT, was a proadipogenic miRNA. Further study demonstrated that miR-450a-5p promoted adipogenesis through repressing expression of WISP2 by targeting its 3′ untranslated region. Additionally, Exo-AT could also downregulate the expression of WISP2, while miR-450a-5p inhibitor reversed this effect. Moreover, inhibition of miR-450a-5p impaired adipogenesis mediated by exosome-like vesicles. In conclusion, Exo-AT mediates adipogenic differentiation through a mechanism involving transfer of miR-450a-5p.

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As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.