Wide-field macroscopic fluorescence lifetime imaging for pre-clinical assessment of targeted drug delivery

Abstract

This study optimized our wide-field MFLI FRET imaging for preclinical applications by selecting the best near-infrared (NIR) FRET pair and implementing reflectance imaging geometry. Comparison of five NIR FRET pairs using a cell-based assay revealed that Alexa Fluor 700 and Alexa Fluor 750 was the best NIR pair due to its brightness and a good relationship between FRET level and acceptor to donor ratios. Implementation of reflectance illumination geometry allowed whole-body imaging of small animals. The validation was performed with the previously implemented transmission illumination geometry. There was no difference between lifetime-based FRET quantification in vitro and in vivo.