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

Sez-Jade K. Chen; Alena Rudkouskaya; Joseph Mazurkiewicz; Marien Ochoa Mendoza; Nattawut Sinsuebphon; Margarida Barroso; Xavier Intes

doi:10.1117/12.2508056

4 March 2019 IRDye QC-1 as a near-infrared dark acceptor for fluorescence lifetime FRET measurements in cells and in live intact animals (Conference Presentation)

Sez-Jade K. Chen, Alena Rudkouskaya, Joseph Mazurkiewicz, Marien Ochoa Mendoza, Nattawut Sinsuebphon, Margarida Barroso, Xavier Intes

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Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108820N (2019) https://doi.org/10.1117/12.2508056
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

Fluorescence lifetime imaging (FLI) is widely regarded as the most robust means to utilize Förster 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.

Conference Presentation

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Sez-Jade K. Chen, Alena Rudkouskaya, Joseph Mazurkiewicz, Marien Ochoa Mendoza, Nattawut Sinsuebphon, Margarida Barroso, and Xavier Intes "IRDye QC-1 as a near-infrared dark acceptor for fluorescence lifetime FRET measurements in cells and in live intact animals (Conference Presentation)", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820N (4 March 2019); https://doi.org/10.1117/12.2508056

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KEYWORDS

Fluorescence resonance energy transfer

Luminescence

In vivo imaging

Near infrared

Confocal microscopy

Multiplexing

Absorbance

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