Cotroneo, E. R. | bioRxiv Science

bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗

bioRxiv2024

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, revealed using primary antibodies (1.Abs) 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 1.Abs revealed by engineered secondary nanobodies (2.Nbs) that allow to selectively erase the fluorescence signals. We developed 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.

neuroscience↗