【作者】 刘鑫；

【导师】 郑国锠； 刘恒；

【作者基本信息】 兰州大学 ， 细胞生物学， 2009， 博士

【摘要】 双光子激光共聚焦荧光显微镜（Two-photon laser scanning fluorescencemicroscopy,TPM）因其具有独特的优势而被广泛应用于生命科学及医学领域。然而由于缺乏真正意义的双光子荧光探针,使双光子荧光显微镜的应用受到了很大程度上的限制。利用双光子荧光显微镜,长时间高空间分辨地活体观测核酸的生命活动是非常有意义的工作,然而目前尚无同时满足优良双光子特性和活体染色要求的双光子核酸荧光探针被报道。本论文针对两种以咔唑荧光团为母体结构的双光子荧光探针BMVC和9E-BHVC开展工作。我们发现,在溶液中该咔唑系列荧光探针对环境变化具有敏感的响应能力:溶剂极性和局部微环境粘度的改变都能在很大程度上影响探针的荧光发射,此外探针在缓冲液中仅有微弱的荧光,但与DNA作用后荧光显著增强,据此我们推断该探针是能形成TICT态的一类0-1逻辑的分子光开关型探针。BMVC和9E-BHVC在DNA中有较高的量子效率（23.6%和38.9%）和大的活性吸收截面（45.08 GM和90.64 GM）,且对DNA具有高的亲和力(结合常数分别为5.2×10⁶M（-1）和7.8×10⁶M^-1)。与BMVC相比,9E-BHVC表现出作为双光子荧光探针更好的特性,例如具有更大的活性吸收截面和更高的结合常数,且具有更好的水溶性（可达到mM级）。9E-BHVC这些优良的性质预示着该分子如果能够在复杂的细胞环境中特异性地识别DNA,那么它将是真正意义的双光子核成像探针。因此,本论文进一步对9E-BHVC的细胞核成像能力作了详尽的研究:研究发现9E-BHVC具有广范的适用性和光稳定性,能够特异的明亮的成像细胞核,而在细胞质中几乎没有荧光。对于固定后的动物细胞染色需要的浓度极低,染色时间很短,且在高浓度长时间的染色条件下能够着染活动物细胞。双染实验的结果证实,9E-BHVC在固定细胞和活细胞上染色细胞核的数量与经典的核酸染料（DAPI）表现出很好的一致性,因此,我们可以说:“9E-BHVC探针是和经典的商业单光子荧光探针DAPI具有相似染色能力的细胞膜半通透性的双光子核酸荧光探针”。本论文的意义在于为生命科学工作者们提供了一个细胞膜半通透性的、真正意义上的双光子核酸荧光探针。更多还原

【Abstract】 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.更多还原