NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals

Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, which have been revealed using primary antibodies and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional antibodies revealed by engineered secondary nanobodies that allow the selective removal of fluorescence signals. We develop three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5–8 targets for d STORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays. Confocal and super-resolution tools used for multiplexed fluorescence imaging often demand specialized equipment and software. Here, the authors present NanoPlex, a universal method for high-plex labeling while preserving cellular ultrastructure. The approach relies on engineered secondary nanobodies that allow the selective removal of fluorescence signals.