【作者】 张元红；

【导师】 黄柏标； 于晓强；

【作者基本信息】 山东大学 ， 材料学， 2010， 博士

【摘要】 双光子荧光显微镜是一种强有力的研究动力学过程、活细胞成像特别是活组织生命现象的工具。双光子显微镜在成像细胞和组织方面具有以下几个优点：固有的局域激发,大的穿透深度(>500μm),低的自发荧光和自吸收,小的光损伤和光漂白,这使得长时间深入成像完整组织而不会引起组织破环成为可能。然而大多数用于双光子荧光显微镜的单光子荧光探针由于自身的双光子荧光活性吸收截面小而限制了它们在双光子显微镜上的应用。因此,发展具有大活性吸收截面的双光子荧光探针是非常必要的。本论文合成了系列咔唑衍生物类双光子荧光核酸探针和酸性囊泡探针,结果表明它们具有大的双光子荧光活性吸收截面,并且能够较好地进行双光子荧光活细胞或者活组织成像。1.咔唑阳离子衍生物在生物成像中的应用：在不同激发波长条件下,咔唑阳离子衍生物9E-BHVC、9E-BMVC和BMVC在不同溶剂中的双光子活性吸收截面均比传统的核酸探针DAPI的大。特别是在DNA存在时,衍生物的双光子荧光活性吸收截面达到了45-96 GM,而DAPI的仅为2.18 GM。随着DNA的增加,衍生物的紫外吸光度先下降后上升,吸收峰位红移；在350-550 nm范围内出现诱导圆二色信号；单光子荧光强度较其在缓冲溶液中的荧光强度分别增加了485(9E-BHVC)、83(9E-BMVC)和374(BMVC)倍,其结合常数达到了106-107量级。在双光子激发下,衍生物同样表现出对DNA的灵敏响应,双光子荧光强度显著增加,较其在缓冲溶液中的强度增大了36-54倍。对咔唑衍生物进行了生物学性能的评估：以双光子激光器的最佳激发波长800nm进行激发,获得了拟南芥活细胞和活组织中核酸的照片；以740 nn为激发波长,进行了拟南芥活组织的成像。当激发波长为800 nm时,咔唑衍生物和DAPI双染的原生质体实验结果表明,衍生物的染色细胞核的位置和染色细胞核的数目均与DAPI一致。9E-BHVC在成像拟南芥活组织中时,表现出了较其它两个衍生物更优越的染色性能。着重考察了9E-BHVC在拟南芥活组织中的成像性能：在相同的激发光能量下记录了9E-BHVC和DAPI荧光强度随成像深度的变化,在同一个成像深度上,9E-BHVC的荧光强度要比DAPI的大；在相同的成像深度条件下测定了9E-BHVC和DAPI荧光强度随激发光能量的变化,在同一个激发光能量条件下9E-BHVC的荧光总是比DAPI的强。结果表明,利用9E-BHVC可以获得更深入的双光子荧光图像或者在更低的激发光能量下可以获得满意的双光子荧光图像。当激发波长为740 nm时,9E-BHVC在成像浑浊组织方面也比DAPI有优势。9E-BHVC是一个有效的双光子荧光核酸探针,具有成为商业双光子荧光探针的潜力：(i)在DNA存在下有大的活性吸收截面(90 GM)和在缓冲溶液中小的活性吸收截面(2.4 GM)；(ii)大的结合常数(1.02×107 M-1)；(iii)好的DNA"光开关”性能；(iv)像DAPI一样,能够专一的全部的染色活植物组织中的细胞核,但比DAPI染的深；(v)具有高的光稳定性和低的生物毒性。2.咔唑阳离子衍生物与DNA相互作用的光谱研究：利用滴定实验,考察了咔唑阳离子衍生物BHVC-DNA体系的紫外光谱、圆二色光谱、单光子荧光光谱和双光子荧光光谱的变化,从而对BHVC和DNA的结合方式进行了探讨。当以磷酸根的浓度代表DNA时,这些光谱中均存在一个比值([磷酸根]/[BHVC]=2～4.3):当小于此值时, BHVC的吸光度减小、在320-550nm范围内出现负的诱导圆二色信号、DNA在275 nm处的圆二色信号由正变负、单／双光子荧光峰红移、BHVC在热变性核酸中的荧光强度较其在未变性核酸中的大；当大于此值时,BHVC的吸光度增加、在320-550 nm范围内出现负/正模式的诱导圆二色信号、DNA在275 nm处的圆二色信号强度降低、单/双光子荧光峰蓝移、BHVC在热变性核酸中的荧光强度较其在未变性核酸中的小。除此以外,在高比例和低比例的DNA-BHVC体系中加入KI时,两个体系均表现出荧光淬灭。可以推断：BHVC在低比例条件下主要以插入方式与DNA结合,咔唑部分插入到碱基对之间而吡啶部分暴露在外；BHVC在高比例时主要是以沟槽结合方式结合到DNA链外部。3.酸性环境敏感的咔唑衍生物的性能评估：咔唑中性分子衍生物(9E-BVC)在乙醇-三氟乙酸体系中的双光子荧光活性吸收截面可达到48.1 GM,远大于其在相应激发波长条件下乙醇中的活性吸收截面数值2.9 GM。在乙醇-三氟乙酸体系中的双光子荧光发射峰位为595 nm,较其在乙醇体系中的发射峰位(470 nm)红移了125 nm。并且可以在双光子激发条件下成像活细胞中的酸性囊泡。本文对所合成的系列核酸探针在浑浊组织中的成像性能做了详细评估,并对这类咔唑阳离子衍生物与DNA的结合方式进行了探讨；研究了具有强的荧光发射强度和大的斯托克斯位移的双光子酸性囊泡探针分子的成像性能。这为双光子荧光探针的设计、合成提供了依据,将进一步推动双光子荧光显微镜在生物学领域的应用。更多还原

【Abstract】 Two-photon fluorescence microscopy (TPM) has been proved to be a powerful technique for the study of dynamic processes, live-cell imaging and especially living phenomena in living tissues. It offers several advantages for imaging of cells and tissues: deeper penetration depth (>500μm), lower tissue autofluorescence and self absorption, and reduced photodamage and photoleaching, in addition to the intrinsically locallized excitation. This allows imaging deep inside the intact tissue for a long period of time without tissue preparation artifacts. However, most of the one-photon fluorescent probes used for TPM have low two-photon excited fluorescent action cross section(φ×δ) that limit their use in TPM. Therefore, there is a pressing need to develop efficient two-photon fluorescent probes with largerφ×δfor in vivo imaging. In this thesis, series of crabazole derivatives type of two-photon fluorescent nucleic acid and acidic vesicles probe were synthesized, they have optimized two-photon properties and could image living cells and/or living tissues by TPM.1. Two-photon fluorescence imaging of nucleic acid in living plant turbid tissue with dicationic carbazole derivatives:At every excitation wavelength, the values ofφ×δof dicationic carbazole derivatives(9E-BHVC,9E-BMVC and BMVC) in various solvents are larger than that of DAPI. It is worth to note that theφ×δof 9E-BHVC,9E-BMVC and BMVC are in the range of 45-96 GM in the presence of DNA, whileφ×δof DAPI is 2.18 GM. With addition of DNA, electronic absorption spectra of the three molecules show significant bathochromic shifts (20-23 nm) and large absorbance changes in opposite directions; introduced circle dichroism in the range of 350-550 nm are found; one-photon fluorescent intensities are strongly enhanced, with exaltation of 485 (9E-BHVC), 83(9E-BMVC) and 374(BMVC); the binding constants reach 106-107 order. With the two-photon excitation, carbazole derivtives also display sensitive response to DNA, and their two-photon fluorescence exaltation are 36-54 fold. Their imaging DNA ability in living cells and living tissues at the excitation wavelength of 800 nm and 740 nm were envalued. At 800 nm, carbazole derivatives can stain the DNA in protoplasts and the positions and regions stained are consistent with those of DAPI. However, when staining living tissue of Arabidopsis thaliana, the abilities of 9E-BHVC and 9E-BMVC are superior to BMVC, and 9E-BHVC are best. Therefore, the imaging performance of 9E-BHVC in imaging highly scattering specimens were studies in detail. A series of doublestaining microscopic photos of DAPI and 9E-BHVC under the same imaging conditions and at different depths were obtained. In the same imaging depth, the fluorescent intnesity of 9E-BHVC are stronger than that of DAPI. Similarity, at the same power, the fluorescent intnesity of 9E-BHVC are also more intensitive than that of DAPI. Experimental results show:compared to DAPI,9E-BHVC can be used to carry out deeper observation using the same incident power, or can be used to obtain usable fluorescent images by using a lower incident power. At 740 nm,9E-BHVC also exhibits better imaging ability in living tissues than that of DAPI.Thus, we can say that 9E-BHVC is an efficient two-photon fluorescence nucleic acid probe. It possesses potential to become a commercial two-photon fluorescent DNA probe:(i) high (i)highφ×δwhen binding to DNA (90 GM) and lowφ×δin buffer solution in the absence of DNA (2.4 GM), (ii) large intrinsic binding constant to DNA (1.02×l07 M-1), (iii) good DNA "light-switch" properties, (iv) exclusively and fully labeling nuclei in living plant tissue same as DAPI, especially deeper imaging than DAPI. (v) high photostability and low biological toxicity. These excellent properties make 9E-BHVC important value to biologist for significant biological discovery.2. Analysis of interaction between dicationic carbazole derivatives and DNA by spectra:Using electronic absorption spectra, circle dichroism spectra and one-and two-photon emission spectra, the binding modes between BHVC and DNA have been studied. All spectral show such ratio of [phosphate of DNA]/[BHVC] in the range of 2～4.3. When less than the ratio, the absorbance of BHVC decrease, negative introduced circle dichroism in 320-550 nm are observed, positive circle dichroism signal of DNA at 275 nm change to be negative, peak position of one-and two-photon spectra red shift, fluorescence intensities of BHVC in the presence of denatured DNA are higher than that in normal DNA. When larger than the ratio, the absorbance of BHVC increase, negative/positive mode introduced circle dichroism in 320-550 nm are observed, intensity of positive circle dichroism signal of DNA at 275 nm decrease, peak position of one-and two-photon spectra blue shift, fluorescence intensities of BHVC in the presence of denatured DNA are lower than that in normal DNA. In addition, whether in low ratio of [phosphate]/[BHVC] or high ratio of [phosphate]/[BHVC] system, the one-photon fluorescence intensity of BHVC decrease with addition of KI. Therefore, BHVC bind to DNA with carbazole motif intercalate into base pair of DNA and pyridium motif exposed outside at low ratio, while BHVC bind to DNA with aggregation on the surface of DNA at high ratio.3. Basic research on properties of carbazole derivatives sensitive to acid enviroment:TheΦ×δof the title compound (9E-BVC) in acidic (0.1 M trifluoroacetic acid, TFA) ethanolic solution reach 48.1 GM, while at the same excitation wavelength, theΦ×δin ethanol only 2.9 GM. On the other hand, the two-photon fluorescent peak position in acidic (0.1 M trifluoroacetic acid, TFA) ethanolic solution is 595 nm, while that in ethanol only 470 nm. Moreover,9E-BVC can stain acidic vesicles in living cell by TPM.The imaging ablitiy of DNA probe in living turbid tissues are envalued in detail and the binding modes are disussed; Besides, a basic research on imaging ability of two-photon fluorescence acidic vesicles probe which possess both greatly enhanced emission intensity and large stokes shift, has been carried out. These provide guidance for the design and synthesis of two-photon fluorescence probe and will further promote the application of TPM in biological field.更多还原