Preclinical imaging using near-infrared (NIR) fluorescent markers is a non-invasive approach to molecular imaging with many applications ranging from drug delivery monitoring to immune cell tracking and image-guided surgery. It is however limited by signal absorption and scattering within tissues, which make it difficult to localize and quantify signal sources. Moreover, broad absorption and emission spectra make it difficult to use multiple NIR dyes for multiplexed imaging. Fluorescence lifetime imaging (FLI) helps solve some of these problems by providing a contrast mechanism that is sensitive to the intracellular environment but not to signal intensity and can separate dyes with similar spectra but different lifetimes. So far, however, in vivo FLI has found few applications due to the complexity of standard FLI data acquisition and analysis. Here we show that in vivo wide-field time-gated macroscopic FLI (MFLI) imaging, combined with phasor analysis of NIR fluorescence lifetime data, addresses most of these issues. We illustrate these capabilities with a study of in vivo transferrin ligand-receptor engagement dynamic in anesthetized mice using Förster resonant energy transfer (FRET), and validate our results by comparing them with standard lifetime fitting. Our approach speeds up data analysis by several orders of magnitude and requires less data, while providing easy-to-use and interpret data visualization, opening up the possibility of real-time in vivo MFLI of fast dynamic processes.
Sez-Jade Chen, Nattawut Sinsuebphon, Alena Rudkouskaya, Margarida Barroso, Xavier Intes, and Xavier Michalet, "Fast in vivo quantification of receptor engagement by phasor analysis of NIR fluorescence lifetime (Conference Presentation)," Proc. SPIE 10487, Multimodal Biomedical Imaging XIII, 104870I (Presented at SPIE BiOS: January 28, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2289084.5751458205001.
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