Lifetime-engineered ruby nanoparticles (Tau-Rubies) for multiplexed imaging of μ-opioid receptors

To address the growing demand for simultaneous imaging of multiple biomarkers in highly scattering media such as organotypic cell cultures, we introduce a new type of photoluminescent nanomaterial termed "tau-ruby"composed of ruby nanocrystals (Al₂O₃:Cr³⁺) with tunable emission lifetime. The lifetime tuning range from 2.4 to 3.2 ms was achieved by varying the Cr³⁺ dopant concentration from 0.8% to 0.2%, affording facile implementation of background-free detection. We developed inexpensive scalable production of tau-ruby characterized by bright emission, narrow spectrum (693 ± 2 nm), and virtually unlimited photostability upon excitation with affordable excitation/detection sources, noncytotoxic and insensitive to microenvironmental fluctuations. By functionalizing the surface of tau-rubies with targeting antibodies, we obtained different biomarkers suitable for multiplexed lifetime imaging. As a proof of principle, three tau-ruby bioprobes, characterized by three mean lifetimes, were deployed to label three μ-opioid receptor species expressed on transfected cancer cells, each fused to a unique epitope, so that three types of cells were lifetime-encoded. Robust decoding of photoluminescent signals that report on each cell type was achieved by using a home-built lifetime imaging system and resulted in high-contrast multiplexed lifetime imaging of the cells.