Near-Infrared Organic Structures with Extended Lifetimes to Unlock the Potential of In Vivo Fluorescence Lifetime Imaging

Abstract In vivo fluorescence lifetime imaging (FLI) is an advanced optical imaging modality with the capability to reveal additional biological information compared to fluorescence intensity (FI) imaging. In addition to enabling tissue discrimination based on microenvironmental variations, lifetime contrast allows the differentiation of spectrally overlapping fluorophores, thereby expanding the high potential of multiplexing imaging. Until now, most of the studies evaluating the benefits of in vivo FLI in terms of enhanced sensitivity and specificity for tumor imaging with fluorescent contrast agents have been conducted using heptamethine cyanine dyes. While these fluorophores are highly successful for conventional in vivo fluorescence imaging, they are characterized by short singlet excited-state lifetimes (generally below 1 ns), making it challenging to achieve satisfying tissue contrast and to implement multiplexing imaging strategies based on their lifetime differences. Here, we report a bioconjugatable, water-soluble NIR-emissive aza-BODIPY dye with a 100% longer lifetime than IRDye 800CW in physiological conditions. We demonstrate its suitability for in vivo FLI when conjugated to cetuximab and reveal how conjugation methods impact the lifetime characteristics of fluorescent antibody conjugates.