Two–photon excitable red fluorophores for imaging living cells

Abstract

We report two novel long-wavelength ICT fluorophores (compound N1-1 and N1-2) derivative from the core structure of 1-oxo-1H-phenalene-2,3-dicarbonitrile (OPDCN). They exhibit bright fluorescence, reasonable two-photon activity and sufficient water-solubility. The synthesis of compound N1-1 and N1-2 is OSNArH (oxidative nucleophilic substitution of aromatic hydrogen), which is very facile and versatile. Owing to the hydrophilic side chains, the water-solubility of N1-1 and N1-2 (11 μM and 12 μM, respectively) are sufficient for cell imaging. N1-1 and N1-2 are more easily to diffuse into living cells than the reference compound N1-3 with poor water solubility. Moreover, compound N1-1, possessing an electron-donating morpholino unit sensitive to pH, exhibits the dependence of fluorescence on pH in the range from 3 to 10, with a pKa at around 5.5. Thus, N1-1 is suitable for imaging lysosomes (acidic organelles) in living cells. Through the technique of femtosecond two-photon excitation fluorescence (TPEF), two-photon activity of Compound N1-1 and N1-2 are evaluated to be appropriate for two-photo microscopy (TPM) imaging. Finally, N1-1 is applied to image lysosomes of live cells under TPM. It is worth mentioning that the test on TPEF activities of OPDCN fluorophore family is performed for the first time. And N1-1 is the first subcellular organelle-targetable probe from this family that is practically applied in TPM imaging.