In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short-lifetime near-infrared dyes and time-gated imaging.
FRET
binding equilibrium
fluorescence
in vivo
lifetime
mouse
near-infrared
pharmacokinetics
phasor
transferrin
Journal
Journal of biophotonics
ISSN: 1864-0648
Titre abrégé: J Biophotonics
Pays: Germany
ID NLM: 101318567
Informations de publication
Date de publication:
03 2019
03 2019
Historique:
received:
15
05
2018
revised:
08
10
2018
accepted:
11
11
2018
pubmed:
14
11
2018
medline:
28
5
2020
entrez:
14
11
2018
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30421551
doi: 10.1002/jbio.201800185
pmc: PMC6559731
mid: NIHMS1011451
doi:
Substances chimiques
Coloring Agents
0
Immunoglobulin G
0
Receptors, Transferrin
0
Transferrin
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e201800185Subventions
Organisme : NIBIB NIH HHS
ID : R01 EB019443
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA207725
Pays : United States
Informations de copyright
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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