In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

• Published in: Journal of biophotonics 2019-03, Vol.12 (3), p.e201800185-n/a
• Main Authors: Chen, Sez‐Jade, Sinsuebphon, Nattawut, Rudkouskaya, Alena, Barroso, Margarida, Intes, Xavier, Michalet, Xavier
• Format: Article
• Language: English
• Subjects: Animals; Binding; binding equilibrium; Cell Line, Tumor; Chemical compounds; Coloring Agents - pharmacokinetics; Complex systems; Data processing; Dyes; Energy transfer; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorophores; FRET; Humans; Imaging; Immunoglobulin G - chemistry; in vivo; In vivo methods and tests; Infrared analysis; Infrared Rays; Kinetics; lifetime; Medical imaging; Mice; mouse; near‐infrared; Optical Imaging; Organs; Pharmacokinetics; Pharmacology; phasor; Phasors; Pulsed lasers; Receptors, Transferrin - metabolism; Stoichiometry; Surgery; Tissue Distribution; Transferrin; Transferrin - chemistry; Transferrin - metabolism; Transferrins
• ISSN: 1864-063X; 1864-0648; 1864-0648
• DOI: 10.1002/jbio.201800185

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography. A new application of the phasor approach to analyze time‐gated near‐infrared (NIR) fluorescence lifetime data in vivo is described. The technique is first benchmarked in vitro, using NIR dyes with short lifetimes, and applied to Förster resonance energy transfer analysis of binding equilibria. Application to the study of the kinetics of transferrin‐transferrin receptor engagement in vivo demonstrates both the simplicity and quantitative aspects of this technique.