Synthesis, Spectral Properties And Application Of Two-photon Fluorescent Dyes Based On The Derivatives Of Fluorene And Pyrene

In the1990s, the two-photon fluorescence microscopy led to a revolution in the field of in vivo high-resolution micro-imaging technology. Compared with the traditional single-photon confocal laser scanning fluorescence microscopy imaging technique, the two-photon fluorescence microscopy imaging technology displayed some significant advantages including near-infrared excitation, dark-field imaging, low photobleaching and phototoxicity, target excitation, high horizontal and vertical resolution. So far, the two-photon fluorescence microscopy imaging technique havs become a molecular imaging technology with the highest temporal and spatial resolution, and its emergence and development greatly promoted the development of various fields of life sciences. However, two-photon excitation efficiency of the common fluorescent probes is very low, greatly restricted the application of two-photon fluorescence microscopy imaging techniques in vivo imaging. Therefore, there is an urgent need that is to design an efficient, specific two-photon fluorescencent probe to address these problems.In the firstl part, this thesis provided an overview of the basic principles of two-photon absorption, compared the differences between single-photon and two-photon absorption processes, pointed out the relationship of the materials’structure and their two-photon properties, further discussed the unique advantages of two-photon biological fluorescence microscopy and summarized two-photon fluorescent probes that have already been applicated in biological imaging.The second and third part focused on the experimental study.On one hand, five two-photon fluorescent dyes with D-π-A structure were designed and synthesized. Taking9,9-dimethyl-2-nitro-fluorene as raw materials, these dyes were obtained after the reduction of nitro, substitution of amino, Friedel-Crafts acylation and click reaction. Then their photophysical properties were tested by both single and two-photon spectroscopy.Take TPFLs Dyes for example, it exhibites special properties as follows: solvatochromism, appropriate water solubility, low pH sensitivity, low toxicity and TP action cross section values of232GM in DMSO (TPFL-Lyso). Meanwhile, TPFLs dyes show good cell permeability. TPFL-Lyso can detect lysosomes in live cells and it is clearly capable of visualizing the lysosomes at depths of220μm in live tissues by TPM. Also, TPFL-ER can serve as a TP probe for endoplasmic reticulum. On the other hand, four TP dyes based on pyrene derivatives were synthesized through nucleophilic substitution reaction and Sonogashira coupling reaction. All these dyes show solvatochromism effect to some extent. The TP action cross section values of s-TPPyO, s-TPPy2and TPPy are43GM (THF,800nm),36of GM (EtOH,810nm) and936GM (THF,740nm) respectively determined by the fluorescence method. Thus s-TPPyO, s-TPPy2and TPPy have good cell permeability and application in two-photon fluorescence imaging.