| Recent years, as the prevalence of commercial two-photon microscopy in biological laboratory, the research about the organic TPA materials and their applications in bioimaging has attracted great attention from domestic and foreign scholars and made great progress. Compared with common and confocal microscopy, two-photon fluorescence microscopy (TPFM) offers a number of advantages, including improved resolution of three-dimension imaging, increased penetration depth and decreased photodamage. TPFM satisfied the demand of biologists in monitoring life phenomenon on alive specimen real-timely and dynamically. But the research on two-photon fluorescent probe is lag behind, the wide application of TPM in biology is limited. So finding two-photon fluorescent probe with large two-photon excited fluorescence action cross-section, high fluorescence quantum yield and sensitivity has important theoretical and practical significance. However, rare TPEF turn-on acidic pH probes for imaging intracellular acidic compartments were reported. Therefore TPEF turn-on acidic pH probes with good membrane permeability, high selectivity, suitable pka vaule and a significant TPEF turn-on character for imaging intracellular acidic compartments are still extremely needed.Intracellular pH value plays an important role in intracellular physiological activities such as cell growth, enzyme activity regulation and ion transport. Studies have found that abnormal pH value is associated with many human diseases. Fluorescence imaging method to detect intracellular pH change has unique advantages, especially ratiometric fluorescent probes, which could eliminate the influence of probe concentration and external environment by a built-in correction of two emission bands, displayed prominent advantages and were intensively studied. However, ratiometric organic small molecule fluorescent probes for intracellular pH value are rare reported.In this thesis, six types of carbazole-based fluorescent probes owning good water solubility and two-photon properties were synthesized by Heck reaction. The synthesis route is simple, easy to operate. The target compounds were characterized by NMR, elemental analysis and mass spectra. In this thesis, we detailedly analyzed the UV absorption, one-photon fluorescence and two-photon fluorescence spectra in various solvent of CAE, CA, CAM1, CAM2, CAEI and CAI calculated the fluorescence quantum yield (Φ), two-photon absorption cross-sections (8) and two-photon excited fluorescence action cross-sections (Φ×δ), finally studied the application of these probes on confincal and two-photon microscopy.At the same time, the properties of CAE had been researched in detailes as a fluorescent probe for intracellular pH. The experimental results show that CAE is a novel one-photon ratiometric and two-photon turn-on acidic pH probe with practical application in confocal microscopy and TPM for imaging intracellular acidic compartments. In near-neutral environment, both one-photon excited fluorescence (SPEF) and TPEF of CAE were weak, reversely both can intensively enhance in acidic surroundings. Standard titration experiments of CAE provided a pKa of5.61which matched pH range of intracellular acidic compartments. And then selectivity of CAE to H+was studied in details:noninterference was exhibited among various cations, anions, amino acids and other bioanalytes. Meanwhile, good photostability was recorded under both one-and two-photon radiation conditions. Furthermore, results of cells imaging experiments suggested that CAE can penetrate membranes and get into a living cell facilely. Very interestingly, CAE could visualize the pH value distribution of acidic compartments in live cells by ratiometric methodology in confocal microscopy. Moreover, the probe own large two-photon absorption property in acidic condition and can be applied in TPM. Additionally, co-stain experiments confirmed that CAE has great counterstain compatibilities with MTR and Hoechst33342. |
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