Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing
Albert, L.; Basak, S.; Koerner, H.; Oleksiievets, N.; Mougios, N.; Cotroneo, E. R.; Frei, M. S.; Enderlein, J.; Broichhagen, J.; Simeth, N. A.; Tsukanov, R.; Opazo, F.
Show abstract
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.
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