Abstract

Abstract Expansion microscopy (ExM) facilitates nanoscale imaging under conventional microscopes, but it frequently encounters challenges such as fluorescence losses, low signal‐to‐noise ratio (SNR), and limited detection throughput. To address these issues, a method of orthogonal DNA self‐assembly‐based ExM (o‐DAExM) platform is developed, which employs hybridization chain reaction instead of conventional fluorescence labeling units, showcasing signal amplification efficacy, enhancement of SNR, and expandable multiplexing capability at any stage of the ExM process. In this work, o‐DAExM has been applied to compare with immunofluorescence‐based ExM for cellular cytoskeleton imaging, and the resolved nanoscale spatial distributions of cytoskeleton show outstanding performance and reliability of o‐DAExM. Furthermore, the study demonstrates the utility of o‐DAExM in accurately revealing exosome heterogeneous information and multiplexed analysis of protein targets in single cells, which provides infinite possibilities in super‐resolution imaging of cells and other samples. Therefore, o‐DAExM offers a straightforward expansion and signal labeling method, highlighting future prospects to study nanoscale structures and functional networks in biological systems.