IRDye QC-1 as a near-infrared dark acceptor for fluorescence lifetime FRET measurements in cells and in live intact animals (Conference Presentation)

Fluorescence lifetime imaging (FLI) is widely regarded as the most robust means to utilize Forster resonance energy transfer (FRET) to study protein-protein interactions. Upon donor excitation, FLI estimates FRET occurrence by determining the reduction of the fluorescence lifetime of the donor when in close proximity (2-10nm) of an acceptor. Recently, macroscopic FLI-FRET (MFLI-FRET) in living mice has been attained by using a near-infrared (NIR)-labeled transferrin (Tf) FRET pair. To harness the potential of multiplexing FLI-FRET in live organisms, it is necessary to employ NIR dark acceptor fluorophores to avoid spectral cross-contamination. IRDye QC-1 (QC-1, LI-COR) is a dark quencher that has a broad absorbance spectrum encompassing the NIR range. Herein, we demonstrate that QC-1 is an effective acceptor for quenching of Alexa Fluor 700 (AF700) via FRET in IgG antibody interactions. Additionally, we characterized the cellular uptake of Tf conjugated to QC-1 using confocal microscopy, NIR FLI microscopy, and wide-field MFLI imaging. The AF700/QC-1 FRET pair exhibits a linear trend in FRET with increasing A:D ratio. In vivo MFLI-FRET imaging was performed under reflectance geometry to compare Tf AF700/AF750 and Tf AF700/QC1 at A:D ratio 2:1 2, 6, and 24h post-injection. FRET was detected in the liver, an important organ for pharmacokinetic studies that shows elevated expression of transferrin receptor (TfR), but not in the bladder, an important organ for drug clearance. Although we observed slightly less FRET using AF700/QC-1 compared to AF700/AF750, both in vitro and in vivo, we found that QC-1 is suitable for FRET imaging and multiplexing approaches.