【作者】 崔景强；

【导师】 彭孝军；

【作者基本信息】 大连理工大学 ， 应用化学， 2010， 博士

【摘要】 随着双光子技术的发展,双光子荧光显微镜在生命科学的研究中已经成为最重要的成像工具。与传统的单光子荧光共聚焦显微镜相比,双光子荧光显微镜具有显著的优势,包括近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等。由于双光子吸收过程与两倍光子能量有关,激发只局限在共焦平面内的极小的区域内。因此近红外的激发光源能透过组织,进行深层成像。用于研究离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面的双光子荧光探针,是实现成像的关键。因此,在生命医学领域的成像中,发展高效的双光子荧光探针仍然迫切需要。本文基于ICT机理设计合成了苯乙烯类双光子锌离子比率荧光分子探针BZn和DZn。在BZn和DZn与锌离子作用过程中,锌离子的配位作用减弱了共轭氮原子对荧光团的供电能力,从而引起了吸收和荧光发射波长都蓝移。BZN-Zn的双光子活性吸收截面(Φδ)与传统的TSQ-Zn相比,增大了约18倍。同时BZn在HKE293细胞中实现了锌离子的比率双光子荧光显微成像。探针DZn能够专一识别锌离子,通过肉眼和荧光变化能够很好的区分镉离子。在830 nm处,DZn-Zn的双光子活性吸收截面(Φδ)为185 GM。与TSQ-Zn相比增大40倍。探针DZn是第一个能够区分锌离子与镉离子的双光子锌离子比率荧光探针,能够在120μm深度下,可视化HEK293活细胞和活体组织内源性锌离子的双光子比率荧光成像。设计合成了一个水溶性的铅离子双光子比率荧光分子探针DCN4。随着铅离子浓度的增加,导致在590 nm处出现新的发射峰并逐渐增强。在740 nm处,DCN4-Pb的双光子活性吸收截面(Φδ)为51 GM。探针DCN4是第一个双光子铅离子比率荧光探针。基于疏水作用设计合成了新的双光子蛋白荧光探针DCN2。DCN2与HSA作用后,荧光光谱蓝移73 nm,量子产率增大90倍。荧光强度随着浓度的增加具有良好的线性关系(R=0.999),直到蛋白浓度为20μM(1320μg ml-1)。利用SDS-PAGE凝胶电泳后的HSA与DCN2染色,然后进行扫描成像分析,探针染色的过程仅需要30 min,而且,无论凝胶洗前和洗后的荧光成像都很好。DCN2-HSA的双光子活性吸收截面(Φδ)为85 GM在830 nm。能够利用双光子显微成像能够在180μm深度下,进行实现了HEK293活细胞和活体组织内蛋白双光子荧光显微成像。更多还原

【Abstract】 With the developments of technologies based on two-photon, two-photon fluorescence microscopy is becoming an essential imaging system in the research on life science. In comparison with one-photon confocal imaging, two-photon fluorescence microscopy can offer many advantages including NIR (near infrared) photons as excitation source, imaging in the black background, avoidance of photodamage and photobleaching, fixed target excitation, high transverse and longitudinal resolutions, small absorption coefficient of light in tissue, lower tissue autofluorescence, etc. As the two-photon (TP) absorption process is quadratically intensity-dependent, the excitation is confined to a small volume in the focal plane. Furthermore, the use of an infrared laser source improves light penetration depth in tissues, thus allowing deeper tissue imaging. Two-photon fluorescent probes applied to studies on the contents of ions and the effects of their contents upon physiology, physiological action mechanisms of ions, interplays between ions and molecules, distributions of specific molecules and their interactions, and so on, play an important role in imaging. Therefore, Two-photon fluorescent probes with superior photophysical properties are still required for imaging of biomedical field.The stilbene TP Zn2+ratiometric fluorescent probes BZn and DZn based on ICT mechanism have been designed and synthesized. The formation of complex BZn-Zn and DZn-Zn weakened the electron-donating character, leading to a blue shift in absorption and fluorescence emission. The TP action spectra of BZN-Zn indicated a 18-fold larger than those of TSQ-Zn. It can also be used in two-photon ratiometric fluorescence microscopy in living HEK293 cells. DZn is selective for over biologically competing alkali-and alkaline-earth-metal ions, and can distinguish Zn2+ from Cd2+ by naked-eye sand fluorimetric experiments. The TP action spectra of DZn-Zn indicated aΦδvalue of ca.185GM at 830 nm,40-fold larger than those of TSQ-Zn. DZn was confirmed to be directly applicable to monitor changes in intracellular Zn2+ in living tissues, owing to its membrane permeability. TP ratiometric imaging with DZN at a depth of 120μm could clearly visualize endogenous Zn2+ in hippocampal slices without suffering from dye localization artifacts.New water-soluble TP ratiometric fluorescent probe DCN4 for Pb2+ containing a 1,4-dicyano-2,5-bis(styryl) benzene fluorophore and 2-(N,N’-bis(carboxylmethyl)) amino-1-carboxylmethoxyl benzene as receptor has been synthesized. With the addition of different concentrations of lead ion, the broad emission intensities at 590 nm gradually increase, and TP action cross section (Φδ) is 51 GM at 740 nm. Novel TP fluorescent probe DCN2 based on hydrophobic effect mechanism has been designed and synthesized to detect HSA (human serum albumin). The formation of complex DCN2-HSA leading to a blue shift of 73 nm in fluorescence emission and an increase of quantum yield for about 90 fold. A good linear relationship was observed up to 20μM(1320μg ml-1) of protein. The staining procedure of DCN2 required only 30 min and fluorescent image of proteins was observed despite the removing of SDS and excess dyes in the gel. The TP action spectra of DCN2-HSA indicated aΦδvalue of ca.85GM at 830 nm TPM (two-photon microscopy) images were obtained from living cells and tissues. Moreover, by using TPM, this probe is capable of monitoring proteins at a depth of about 180μm in living tissues.更多还原