Characterization And Bioimaging Applications Of Two-photon Fluorescent Materials

Two-photon absorption materials have potential applications in the fields of life science and medicine.In the present dissertation,combining the advantages of TPM in biological imaging,we characterized different small organic molecules including pyridinium derivatives as well as metal-complexes to develop target-specific two-photon fluorescent probes.By theoretical and experimental evaluation the structure and properties relationships of the characterized materials were explored in depth.Moreover,the preliminary examination of the two-photon active and biocompatible materials in microenvironment changes and target-specific biological imaging applications using two-photon and super resolution microscopy were performed.The research contents are as follows:1.A series of water-soluble pyridinium derivatives with two-photon absorption in the near infrared region for mitochondria targeting under stimulated emission depletion(STED)nanoscopyConvenient and rational design of small molecular weight,water-soluble,two-photon excited fluorescence(TPEF)probes remains a challenge for practical biological applications.In this study,we designed six novel water-soluble,near infrared TPEF probes(NL1-3,PL1-3),bearing N-methyl pyridinium moiety,which are specifically sensitive to local viscosity.Fluorescence emission bands and two-photon absorption cross-sections of the title compounds showed a significant enhancement with increased solvent viscosity.The compound NL1,a high fluorescent probe that selectively targeted the mitochondria and was sensitive to mitochondrial environment changes,could be applied to differentiate between nystatin treated and normal cell mitochondrial environment.Furthermore,NL1 is membrane potential independent,which was rarely found in contemporary probes.In addition to two-photon confocal microscopy,NL1 also displayed successful mitochondrial staining under stimulated emission depletion(STED)nanoscopy in living cells.In a nutshell,the merits of near infra-red region(NIR)operation,high selectivity,and signal ratio of these probes facilitate the study of mitochondrial targeting,localization processes,and mitochondria microenvironment related diseases.2.A series of two-photon absorption pyridinium sulfonate inner salts targeting endoplasmic reticulum(ER),inducing cellular stress and mitochondria-mediated apoptosis in cancer cells In this study,a two-photon active DiphenthioERl was screened from the four pyridinium sulfonate salt derivatives(TriphenERl-2 and DiphenthioERl-2)for imaging endoplasmic reticulum(ER)and tracking the dynamics of the ER morphology.The photophysical properties of TriphenERl-2 and DiphenthioERl-2 were systemically investigated both experimentally and theoretically,revealing that they possess large Stokes shifts,and large two-photon absorption cross-sections from 163 GM to 2023 GM in the near infrared region using Z-scan method with avoiding spontaneous fluorescence,deep tissue penetration,and low cell damage in living cells.Among them,the compound DiphenthioERl was found to exhibit highest cellular uptake ability and two-photon fluorescence signals under confocal microscopy.DiphenthioERl successfully targeted ER and induced ER-stress,subsequently nuclear misshaping and mitochondria-mediated apoptosis have been displayed.This study thus provides great insights to design novel two-photon fluorescent materials with dual functionality and offer tools to understand ER-stress related mechanism for cell and chemical biologist.3.DiphenthioERl;a two-photon active pyridinium derivative causing opening of blood brain barriers via junctional proteins downregulationDrug delivery to the centeral nervous system(CNS)is one of the major challenges due to the presence of a compact blood brain barrier(BBB),which limits the probability of new CNS drugs only to lipid-soluble compounds with lower molecular weight.In the present study,a small molecular weight,two-photon pyridinium derivative(DiphenthioER1)with good lipophilic properties was applied to an in vitro blood brain barrier model BBB.DiphenthioERl exhibited successful and effective uptake by the bEnd.3 cells and displayed a minute stress at low concentrations and relatively low toxicity upto certain concentrations(?30)?M over 24 hours.Immunofluorescence results showed that bEND.3 cells both in 2D and 3D cultures treated with DiphenthioERl displayed disrupted tight junctions which was prevented in the control group,suggesting that DiphenthioERl causes opening of BBB via down-regulation of tight junction proteins.4.SL-Neu:a two-photon fluorescent chemical probe for NeuN specific live neuron labeling A widely applicable chemical probe for selective staining of live neurons is promptly required to overcome current limitations in live neuron imaging.In the present study,we screened SL-Neu a novel two-photon active fluorescent probe with an exceptional ability to label live neurons specifically over other brain cells.SL-Neu successfully targeted NeuN and proved that NeuN is associated with neuronal RNA.SL-Neu facilitates strong target specific live neuron imaging;its versatility and ease of use sets the foundation for its progress in a numerous neuronal targeting applications.5.Characterization and biological applications of terpyridine Mn2+ complexesThe abundance and low toxicity of manganese have led us to explore the bioimaging applications of manganese complexes.In this study,four terpyridine-manganese complexes(Mnl-Mn4)were characterized and their bioimaging applications were discovered.Based on their photophysical properties,the complex Mn4 amongst them was chosen for further studies as it was giving highest two photon activity as well as uniform particles distribution and morphology.All of the four compounds have extremely low cytotoxicity effect on cell viability over a 24 hours incubation period,which allowed the complexes to be used for biological applications.Strong intracellular fluorescence was perceived in live cell as well as fixed cell imaging by confocal fluorescence microscopy for both normal cells and cancer cells.These results proved that the as-prepared organic Mn-complexes could be applied for cellular imaging with high signal-to-noise ratio.