Design, photophysical properties, and applications of fluorene-based fluorophores in two-photon fluorescence bioimaging: A review

Two-photon fluorescence microscopy (2PFM) emerged as a powerful alternative to conventional one-photon microscopy. 2PFM typically uses two near-infrared (NIR) photons to excite fluorescent dyes, which minimizes light scattering in biological samples. Multiphoton absorption also suppresses background signal and autofluorescence from tissues and allows to achieve higher 3D resolution images with low photodamage and photobleaching. Fluorene dyes possess distinct properties that meet the strict criteria of probes used for 2PFM such as enhanced solubility, photostability, and two-photon absorption cross-section. The fluorene molecule also includes many active positions that allow versatile synthesis, selective functionalization, bioconjugation, and tuning solubility. These properties have led to reporting several fluorene probes including monomers, polymers, and dendrimers with important uses in understanding molecular dynamics and bioimaging. The current review presents a compact summary of fluorene-based fluorophores for 2PFM bioimaging applications, shedding light on structure-photophysical property relationships in fluorenes and polyaromatic probe designs.