Multiplexed Bioimagingvia Lifetime and Spectral Engineeringof Conjugated Oligoelectrolytes

Abstract Multiplexed bioimaging is essential for understanding how distinct organelles coordinate their function in living cells. To increase the number of parameters measurable in parallel, fluorescence lifetime imaging microscopy (FLIM) can be used to complement conventional intensity-based measurements. Conjugated oligoelectrolytes (COEs) represent a promising platform for developing lipid-bilayer vesicle optical probes with a differentiated molecular architecture from frequently used chromophores, yet remain largely unexplored for imaging multiple vesicle populations. Here, we demonstrate how COEs enable multiparametric imaging of live cell intracellular vesicle targets, with compatibility for super-resolution imaging and dynamic monitoring of organelle interactions. The fluorescence lifetime of COEs can be modulated by systematically altering the nature of the intramolecular charge transfer excited states, showcasing molecular design strategies toward developing new donor–acceptor fluorophores. Leveraging this principle, two new COEs with red-shifted emission and longer lifetimes were developed, which complement existing counterparts. We also show that when used alongside commercially available probes, COEs allow one to simultaneously distinguish up to four pairs of spectrally identical probes based on their emission lifetimes. Importantly, the ability to simultaneously label and track multiple distinct intracellular vesicle populations enables rapid, high-content data acquisition and analysis of cellular dynamics. By connecting molecular design and photophysics with advanced imaging techniques, this work suggests a new set of chemical probes that open opportunities for studying biological processes with larger data sets to advance detection methodologies relevant for cell biology and biomedical imaging.