For bioimaging, idealised properties of fluorophores include compact size, facile synthesis, tunability of absorption and emission wavelengths ranging from UV to far IR, large Stokes shift, high quantum yield, and good solubility in aqueous media. Typically, polyaromatic πconjugated fluorophores often suffer from poor solubility due to their propensity to aggregate, and they often involve strenuous multi-step synthesis and purification. Moreover, the presence of a large fluorophore potentially disturbs the properties and biological function of target molecules. Owing to their simpler aromatic skeleton, the design and synthesis of single-benzene-based fluorophores (SBBFs) has received considerable attention. Contrary to large polyaromatic fluorophores, SBBFs contain an electron-donor (D)acceptor (A) type dipolar structure with functional groups incorporated into a compact benzene ring. Due to their facile synthesis, various types of SBBFs have recently been developed and employed for bioimaging applications. In addition, their emission can easily be tuned by varying the substituents on the arene ring. Sun and co-workers reported a simple SBBF precursor tetrafluoroterephthalonitrile (4F-2CN) that was able to efficiently visualise and differentiate the common biological thiols cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) (Figure ). The product from the reaction of 4F-2CN with Cys, 2F-2CN-Cys, displayed two-photon fluorescence properties. Following this, Wang and co-workers designed the γ-glutamyl transpeptidase (GGT) activated watersoluble two-photon fluorescent probe 3F-2CN-GSH. This probe could be cleaved in situ when exposed to GGT-overexpressing cancer cells to form a fluorophore with a 2F-2CN-Cys skeleton (Figure ). Thus, 3F-2CN-GSH has the potential to distinguish cancer cells from normal cells.