The Study Of A Series Of New Two-photon Fluorescent Probes For Nuclear DNA Imaging On Fixed And Living Cells

Two-photon laser scanning fluorescence microscopy(TPM)has been widely used in life science and medicine for its unique features,but the lack of true two-photon fluorescent probes has restricted the extensive applications.Observing the nucleic acid at high-resolution three-dimensional and on time dependence was very significant.However,so far,there were no reports on the probes that exhibit simultaneously the good two photon fluorescent properties and bio-imaging ability of nucleus.Hence,in this dissertation,a series of two-photon fluorophores,BMVC and 9E-BHVC with carbazole as the conjugated center,were studied in detail.We find,in solution,these fluorophores were sensitive to the environment change:the change of solvent polarity and viscosity of local environments would greatly influence the fluorescence emission intensity of BMVC and 9E-BHVC.And the two fluorophores emit weak fluorescence in Tris-HCL buffer,but the intensity of fluorescence increases obviously when bound into DNA.Thus we deduce this possibility that two fluorophores act as light-swtich probes with 0-1 logic due to their TICT.In DNA solution,both BMVC and 9E-BHVC show high quantum efficiency(23.6%and 38.9%,respectively),large active cross sections(45.08GM and 90.64 GM, respectively),and have high affinity to DNA(the binding constants were 5.2×10⁶M^-1 and 7.8×10⁶ M^-1,respectively).Compared to BMVC,9E-BHVC has many better features needed by a two-photon probe,such as larger two-photon action cross sections,stronger affinity to DNA and better water-solubility(mM).These excellent properties of 9E-BHVC all mentioned above imply that it would be a true two-photon nucleic acid fluorescent probe if it can specially recognize the DNA in complex cellulous environment.So this dissertation has investigated the ability of 9E-BHVC on the cell nucleus imaging detaily.The results of these imaging experments show that 9E-BHVC has good photo-stability and wide-adaptability for different kinds of cells.It could image the cell nucleus with bright fluorescence and has low noise in cytoplasm.It was not only be able to image the nucleus of fixed-cell in low concentration with low incident power and also can be used to observe the living cell nucleus at higher concentration and longer incubated time.The double-staining experimental results in fixed and living cells show the amounts of the nucleus marked by 9E-BHVC are entire consistent with those by the classic nucleic acid dye(DAPI),thus we can say:"9E-BHVC can act as the semi-permeability two-photon nucleic acid probe like DAPI."The significance of this dissertation was that it offers to biologists a true semi-permeable two-photon nucleic acid fluorescent probe.