Abstract Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging modality that can provide multiplexed readouts with remarkable sensitivity to cellular microenvironments. Even though fluorescence lifetimes can distinguish fluorophores having overlapping spectral profiles, conventional fluorophores possess a narrow range of emission lifetimes (typically shorter than 5 ns) that limits their potential for multiplexed imaging. In this work, we have systematically designed and evaluated a combination of thermally activated delayed fluorescence (TADF) nanoprobes for multiplexed FLIM. We have synthesized a collection of 36 TADF biocompatible nanoprobes with long and diverse fluorescence lifetimes in aqueous media (up to 15 ns) and employed selected probes for live‐cell imaging of bacterial cells under physiological conditions. By leveraging the exceptionally broad range of fluorescence lifetimes of these TADF emitters, we have achieved unprecedented simultaneous imaging of five nanoprobes within a single spectral window using a FLIM‐phasor strategy. These findings demonstrate that TADF emitters are excellent scaffolds to unlock the capabilities of fluorescence lifetime imaging for multi‐color biological studies.
Cepeda et al. (2026) studied this question.