Au nanodyes as enhanced contrast agents in wide field near infrared fluorescence lifetime imaging

The near-infrared (NIR) range of the electromagnetic (EM) spectrum offers a nearly transparent window for imaging tissue. Despite the significant potential of NIR fluorescence-based imaging, its establishment in basic research and clinical applications remains limited due to the scarcity of fluorescent molecules with absorption and emission properties in the NIR region, especially those suitable for biological applications. In this study, we present a novel approach by combining the widely used IRdye 800NHS fluorophore with gold nanospheres (GNSs) and gold nanorods (GNRs) to create Au nanodyes, with improved quantum yield (QY) and distinct lifetimes. These nanodyes exhibit varying photophysical properties due to the differences in the separation distance between the dye and the gold nanoparticles (GNP). Leveraging a rapid and highly sensitive wide-field fluorescence lifetime imaging (FLI) macroscopic set up, along with phasor based analysis, we introduce multiplexing capabilities for the Au nanodyes. Our approach showcases the ability to differentiate between NIR dyes with very similar, short lifetimes within a single image, using the combination of Au nanodyes and wide-field FLI. Furthermore, we demonstrate the uptake of Au nanodyes by mineral-oil induced plasmacytomas (MOPC315.bm) cells, indicating their potential for in vitro and in vivo applications. Graphical abstract: [Figure not available: see fulltext.]