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

Abstract

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.

Quantification of Trastuzumab–HER2 Engagement In Vitro and In Vivo

“…In this study, the goal was to test TZM–HER2 binding via FLIM-FRET imaging in vitro. In AU565 cells, donor TZM–AF700, in the absence of acceptor TZM–AF750, shows average lifetime values (≈1–1.1 ns), as shown previously for other NIR FRET probes [ 19 , 36 ], whereas in the presence of an acceptor, a significantly reduced lifetime is detected, indicating the occurrence of FRET due to TZM–HER2 interactions. Of importance, in AU565 cells, we found the shortest lifetime species at the plasma membrane, which may be due to the extensive HER2 clustering at the cell surface [ 37 , 38 , 39 ].…”

Wide-field time-gated SPAD imager for phasor-based FLIM applications

“…A separate Cy3B sample was used as the reference dye for phasor calibration, using a τ = 2.5 ns (value measured by TCSPC). µ is the initial dye concentration ratio and χ is the product of the extinction coefficient ratio and quantum yield ratio for both dyes (23). 10 µL of a stock solution of this QD sample (concentration ~ 1 µM) was left to dry on a coverslip and imaged in ambient conditions using the same setup as used in the previous measurements.…”

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

“…In contrast, as expected, the urinary bladder showed very low levels of FD%, suggesting that fluorescence signal is due to excreted Tf-AF700 degradation products. [22][23][24] Although successful, this study was performed using a sequential imaging protocol that increases workload, can lead to some registration errors 8 between the spatial mapping of the biomarkers, and may introduce biological bias due to the dynamic nature of tumor biology. Therefore, we investigated the potential of using dark quencher QC-1 as an alternative FRET acceptor to enable multiplexed imaging in the same animal and concurrent imaging session (Suppl.…”

“…18,21 Recently, our group has pioneered Near-Infrared (NIR) Macroscopic FLI-FRET (MFLI-FRET) to quantitatively monitor ligand-target engagement, a critical component of targeted drug delivery, in live and intact animals. [22][23][24] Using 4 the transferrin (Tf)-transferrin receptor (TfR) system as a biological model, we demonstrated that MFLI-FRET quantitatively reported on intracellular delivery of the drug carrier ligand, providing a non-invasive direct readout of the true payload delivered to pathological cells in live intact animals. 22 Hence, MFLI-FRET is poised to become a mainstream analytical tool to assess target engagement of the ligand-or antibody to their respective receptors in preclinical studies.…”