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

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.