Two-Photon Fluorescent Probes for Bioimaging

With the extensive use of two-photon fluorescence microscopy (2PFM) in the biomedical field, the need for development of fluorescent probes with improved two-photon fluorescence (2PF) properties has triggered extensive studies in the synthesis of new probes that undergo efficient two-photon absorption (2PA). In order to provide a more comprehensive comparison of fluorophores for 2PF bioimaging, a figure of merit (FM) was developed by normalizing the 2PA action cross-section, a commonly used parameter for characterizing bioimaging 2PF probes, by the photodecomposition quantum yield. Another important aspect of developing 2PA fluorophores is hydrophilicity. Although hydrophilic fluorophores are generally preferred in 2PFM bioimaging, hydrophobic fluorophores are typically easier to synthesize and purify, and have been used successfully in 2PFM bioimaging. The methodologies of dispersing hydrophobic fluorophores into aqueous media, such as in a DMSO/water mixture, micelles, silica nanoparticles, or forming polymer nanoparticles, are reviewed. The design and synthesis of hydrophilic 2PA fluorophores, achieved by introducing polyethylene glycol (PEG), anionic acid groups, cationic ammonium salt, and PAMAM dendrimers, is presented. Introduction of specificity to target certain biomarkers via labeling of antibodies, DNA, smallbioactive molecules, and peptides, and for the sensing of sepcific cations and pH, is also reviewed.