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 and Felipe Opazo ()
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Nikolaos Mougios: University Medical Center Göttingen
Elena R. Cotroneo: University of Göttingen
Nils Imse: University of Göttingen
Jonas Setzke: University of Göttingen Medical Center
Silvio O. Rizzoli: University Medical Center Göttingen
Nadja A. Simeth: University of Göttingen
Roman Tsukanov: Georg August University
Felipe Opazo: University Medical Center Göttingen

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-53030-w

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