Photophysics of Conjugated Oligoelectrolytes Relevant to Two‐Photon Fluorescence‐Lifetime Imaging Microscopy

Conjugated oligoelectrolytes (COEs) comprise a class of cell‐membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self‐assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6‐ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE‐S6), and its optical properties within mammalian cells are subsequently studied. Two‐photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE‐S6 internalization via endocytosis. The fluorescence lifetime of COE‐S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.