Label‐Retention Expansion Microscopy (LR‐ExM) for Enhanced Fluorescent Signals using Trifunctional Probes

Abstract Expansion microscopy (ExM) is a super‐resolution imaging technique that bypasses the diffraction limit of conventional optical microscopy (∼250 nm) by enlarging samples with a swellable hydrogel. Combined with various light microscopes, ExM enables an effective resolution ranging from 5 to 70 nm. ExM has now been successfully applied to cell, tissue, and whole‐organism samples, providing biologists with a low‐cost strategy to visualize samples at the molecular level. However, fluorescence signal loss easily happens for beginners and with early versions of ExM protocols. Here, we describe a protocol called label‐retention expansion microscopy (LR‐ExM), which can preserve and enhance the signal of ExM imaging via a series of trifunctional probes. These trifunctional probes are antibody‐based and easy to prepare, and thus suit the needs of most laboratories. © 2024 Wiley Periodicals LLC. Basic Protocol : LR‐ExM using trifunctional probes for enhanced fluorescent signals