Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing

Abstract

Abstract 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.