Nature Communications (Oct 2024)

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

  • Nikolaos Mougios,
  • Elena R. Cotroneo,
  • Nils Imse,
  • Jonas Setzke,
  • Silvio O. Rizzoli,
  • Nadja A. Simeth,
  • Roman Tsukanov,
  • Felipe Opazo

DOI
https://doi.org/10.1038/s41467-024-53030-w
Journal volume & issue
Vol. 15, no. 1
pp. 1 – 17

Abstract

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Abstract 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 dSTORM 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.