An In Vivo Spectral Multiplexing Approach for the Cooperative Imaging of Different Disease-Related Biomarkers with Near-Infrared Fluorescent Förster Resonance Energy Transfer Probes

In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applica- tions, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Forster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies. Methods: We coupled the fluorophores DY-682 (donor; absorption (abs)/ emission (em), 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phago- cytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging sys- tem with multispectral analysis features. Results: When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, un- like the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after inter- nalization of the DY-682-IgG-DY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice. Conclusion: Our simple cooperative-imaging approach ena- bles the noninvasive detection of the presence of two or prin- cipally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interre- lations of target molecules in key pathologic activation pro- cesses such as inflammation, cancer, and neurodegenerative