title: An in vivo spectral multiplexing approach for the cooperative imaging of different disease-related biomarkers with near-infrared fluorescent forster resonance energy transfer probes.
abstract: In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, 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 Förster 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 phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system 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, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization 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.
  
  
  CONCLUSIONS
  Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
authors.affiliation: Institute of Diagnostic and Interventional Radiology, Department of Experimental Radiology, University Hospital Jena, Jena, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Busch
authors.forename: Tom
authors.initials: T
authors.lastname: Schröter
authors.forename: Markus
authors.initials: M
authors.lastname: Grabolle
authors.forename: Matthias
authors.initials: M
authors.lastname: Wenzel
authors.forename: Hanne
authors.initials: H
authors.lastname: Kempe
authors.forename: Werner A
authors.initials: WA
authors.lastname: Kaiser
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
authors.forename: Ingrid
authors.initials: I
authors.lastname: Hilger
mesh_terms: D000327:Adsorption; D000818:Animals; D016477:Artifacts; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004487:Edema / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007259:Infrared Rays; D008264:Macrophages / Q000166:cytology; D008297:Male; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D010587:Phagocytosis; D012867:Skin / Q000737:chemistry / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005456:Fluorescent Dyes