Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.

Fluorescence lifetime (FL) is a robust strategy for multiplexed imaging; however, variability in fluorophores' local microenvironment when used on affinity probes broadens lifetime distributions, limiting its practical applicability. We create a modular platform for FL encoding with nanobody-HaloTag variants, enabling universal FL multiplexing. By characterizing dozens of fluorophores across four HaloTag variants, we generate an FL landscape that reveals tunable, consistent FL signatures within a single spectrum. Leveraging this physicochemically defined encoding, we achieve multiplexed imaging of up to eight targets in a single staining step while preserving high spatial fidelity across cellular and tissue samples. Unlike conventional antibody labeling, our approach decouples probe identity from species constraints and eliminates batch-to-batch variability in FL. These results confirm nanobody-guided FL engineering as a scalable strategy for multiplexed imaging (NanoFLex), offering a standard and straightforward framework for FL multiplexing and expanding the accessible dimensions of optical readout.