Design, Synthesis Of Two-Photon DNA Fluorescent Probes And Their Application In Biological TPM Imaging

There are certain advantages in the processes of two-photon prossess: absorbing longer wavelength but emitting shorter wavelength, high spatial selectivity and high depth of penetration. Upon these features, it provides a powerful tool in the study of biology that W. Denk et al. inventied the two-photon fluorescence microscopy (TPM) in 1990. Because two-photon absorption can take place in any point of three-dimensional space, TPM can be used for biology specimen three-dimensional imaging with high resolution but without confocal pinholes. Due to femtosecond laser with IR output have been used as pumping beam, TPM possesses high depth of penetration without UV-visible photodamage to tissue. However, the lack of two-photon fluorescent probe had limited the development of TPM imaging. In a word, it is very important to study two-photon fluorescent probe with large two-photon action absorption cross sections (Φ×δ) and good biocompatibility.In this thesis, five good amphiphilic molecules with good two-photon properties and well biocompatibility are designed and synthesized. Their photophysical properties are investigated and they are applied in living cell and tissue. The main contents and results are summarized as follows:1. Five carbazole pyridine iodate series organic amphiphilic molecules with large two-photon action absorption cross sections are synthesized utilizing Witting and Heck reaction. ¹HNMR, ¹³CNMR, HRMS and element analysis are utilized for characterization all final compounds. The single crystal of an important organic intermediate 9-Ethyl-3,6-diformyl-9H-carbazole was obtained by using solvent evaporation method and characterized by means of single-crystal X-ray diffraction.2. The liner absorption spectra, one/two-photon emission spectra, fluorescent quantum yield, fluorescent lifetime and two-photon cross sections and action cross actions have been investigated and the solvatochromism has been studied. The relationships between structure and property are discussed in this thesis. Ethyl group covalently linking to the N-atom of the carbazole moiety and the increasing lengths of both ends result in biggerΦ×δand higher quantum yield. There are very large differences in 3,6-/2,7-carbazole derivatives, competitively 2,7- derivatives can give higher quantum yield, longer lifertime and better two-photon properties.3. Fluorescent titration experiments have been carried out in order to determine whether five molecules can be used as two-photon fluorescent DNA probes. In the process of titration ctDNA was used as the model, the liner absorption spectra and one/two-photon emission spectra are obtained by using standerd method. The spectra of BMVC, BMVEC and 9E-BHVC binding ctDNA was mainly analyzed to find the possible mode of binding. Effects of different excitated wavelengths on two-photon fluorescent spectra have been investigated and the optimal excited wavelengths were obtained for TPM imaging. BMVC, BMVEC and 9E-BHVC can be used as "turn-on" fluorescent probes according to fluorescent titration experiments.4. Animal fixed cell, living plant cell and tissue were selected as specimens to investigate the specificity of BMVC, BMVEC and 9E-BHVC in labelling DNA in TPM imaging. Three probes can stain DNA in cell and living tissue, and the positions and regions stained by them are wholly consistent with that of DAPI according to double-staining experiments. Moreover 9E-BHVC and DAPI are selected to stain living tissue, the relationship between flluorescent intensity and detection depth are also investigated in this thesis.