Long-wavelength Emission Two-photon Fluorescent Probes And Their Applications In Imaging Biomolecules

The fluorescence imaging technique has attracted much more attention in applications of biochemistry and medicine,benefiting from its relatively low cost,easy operation,high selectivity,high sensitivity,excellent biocompatibility,and non-invasive real-time dynamic tracking of changes in endogenous detectors.Compared with single-photon fluorescence imaging,two-photon fluorescence imaging provides some crucial merits including weak light scattering,deeper tissue penetration,high spatio-temporal resolution,prolonging observation time and reducing photobleach.Till now,commonly used two-photon fluorophores for two-photon fluorescence imaging are quinoline fluorophore,fluorine fluorophore,carbazole fluorophore,naphthalene fluorophore,naphthalimide fluorophore,rhodamine and fluorescein.Upon being excited with NIR light(700-900 nm),most of them give shortwave emission(< 600 nm).According to previously reported,in complex biological systems,weak light scattering and background fluorescence can be obtained by exciting with long wavelength of light,the optimal wavelength is 900 nm.It is well known that fluorescence spectroscopy includes fluorescence excitation and fluorescence emission.Two-photon excitation can only solve the disadvantages caused by short-wave excitation.To obtain sufficient signal-to-noise ratio and deep tissue imaging depth,fluorescent probes still should possess the characterostics that can supply long-wavelength emission(far-infrared to near-infrared).Unfortunately,the progress for developing far-infrared and near-infrared two-photon fluorophores is significantly retarded due to the complex synthesis procedure,weak solubility and poor selectivity.Therefore,designing and synthesizing two-photon fluorescent probes with long-wavelength emission(> 600 nm)are of great significance for the practical applications of organic small-molecule fluorescent probes in clinical diagnosis.To overcome the limitation of the existing two-photo fluorescent probes which possess short emission wavelength,naphthalimide and tetrahydroxanthone were used as fluorophore to synthesize two-photo fluorescent probes with long emission wavelength.Furthermore,the synthesized two-photo fluorescent probes were successfully used for imaging NO and cysteine,and their relationship with some medicines.The content of this dissertation can be described as following:1.A novel lipid droplet localized two-photon fluorescent probe,TAN,was synthesized using naphthalimide as a fluorophore and extending the ?-conjugate system with triphenylamine.The emission wavelength of TAN red-shifts to 638 nm.TAN shows excellent selectivity towards NO,and thus was encouraged to detect NO with high sensitivity and to image NO with high-resolution.The results demonstrated that TAN possess some superior merits such as excellent selectivity,high light stability,large Stokes shift(188 nm)and large two-photon action section(64 GM).In addition,the tissue imaging depth can reache 167 ?M.Furthrtmore,TAN was also successfully applied for imaging NO produced by chemotherapeutic drugs in lipid droplets in cells and tissues.As a result,visual evidences can be provided for exploring the regulatory function of NO towards the resistance to anti-tumor drugs.2.A D-?-A-?-D mitochondrial targeting NIR two-photo fluorescent probe,TNC,was successfully synthesized by using tetrahydroxanthone as fluorophore.The emission wavelength of TNC can extend to 672 nm.A large two-photon action cross-section of the TNC,157 GM,can be achieved.The water solubility is also significantly improved(PBS/DMSO = 99-5,v/v),and the tissue penetration depth can reache 274 ?M.Furthermore,TNC was successfully applied for detecting and imaging endogenous and exogenous Cys generated from cells.