双光子荧光材料的表征和生物成像的应用

摘要

双光子吸收材料在生命科学和医学领域具有潜在的应用前景。本文对不同的有机小分子（包括吡啶衍生物和金属配合物）进行了研究，设计合成靶向特异性的双光子荧光探针。通过理论计算和实验测试，深入探讨了特征材料的结构和性能关系。此外，使用双光子和超分辨率显微镜对具有双光子活性和生物相容性材料进行了微环境变化和生物靶向性成像研究。研究内容如下:
　　1.双光子吸收的水溶性吡啶鎓衍生物在超分辨率显微镜(STED)下靶向线粒体研究
　　水溶性好的双光子荧光小分子探针的设计仍然是生物成像的面临挑战。在这项研究中，我们设计了六种新颖的水溶性近红外双光子荧光探针(NL1-3，PL1-3)，其中吡啶盐部分对粘度特别敏感。目标化合物的单双光子荧光都随着溶剂粘度增加显着增强。化合物NL1是一种选择性靶向线粒体且对线粒体环境变化敏感的荧光探针，可用于区分制霉菌素处理的和正常细胞的线粒体环境。此外，NL1是膜电位独立的，这种现象很少报道。除了双光子共聚焦显微术外，NL1还在活细胞的STED纳米级显微镜下显示清晰的线粒体染色。总之，这系列生物探针具有近红外(NIR)发光、高选择性和高信噪比，有利于研究线粒体靶向、定位和相关疾病的检测。
　　2.双光子吡啶磺酸内盐靶向内质网(ER),诱导细胞应激和线粒体介导的癌细胞凋亡
　　设计合成系列吡啶磺酸盐衍生物(TriphenER1-2和DiphenthioER1-2)，从实验和理论上系统研究了它们的光物理性质，实验结果显示它们具有较大的斯托克斯位移，Z扫描结果显示它们具有大的双光子吸收截面(从163GM到2023GM)。生物测试发现化合物DiphenthioER1表现出最好的细胞摄取能力和在细胞中具有最高的双光子荧光信号。DiphenthioER1成功靶向内质网应，随后出现核畸形和线粒体介导的细胞凋亡。因此，该研究为设计具有双重功能的新型双光子荧光材料提供了很好的借鉴，并为细胞和化学生物学家提供了理解ER-应激相关机制的探针。
　　3.双光子活性吡啶鎓衍生物通过连接蛋白下调导致血脑屏障开放
　　由于存在紧凑的血脑屏障(BBB)，将药物输送至中枢神经系统(CNS)是重要的挑战，BBB限制CNS药物只能是小分子量的脂溶性化合物。在本研究中，将具有良好亲脂性的小分子量双光子吡啶衍生物(DiphenthioER1)应用于体外血脑屏障模型BBB。DiphenthioER1展示bEnd.3细胞有效摄取，在低浓度下显示微小应激，并且在24小时内在特定浓度(≤30)μM下显示低毒性。免疫荧光结果显示用DiphenthioER1处理的2D和3D培养物中的bEND.3细胞显示出被破坏的紧密连接蛋白(Tight Junctions)。
　　4.SL-Neu:用于NeuN特异性活神经元标记的双光子荧光探针
　　在本研究中，我们筛选了一种新型双光子活性荧光探针SL-Neu，该探针能够特异性标记脑细胞中的活神经元。SL-Neu成功靶向NeuN并证明NeuN与神经元RNA相关。SL-Neu具有特异性靶向活神经元成像，其多功能性和易用性为其在神经元靶向应用中的进展奠定了基础。
　　5.三联吡啶Mn2+配合物的生物研究
　　锰的磁性和低毒性使我们能够探索锰配合物的生物成像应用。在这项研究中，合成了四个三吡啶锰配合物(Mn1-Mn4)并研究了它们的生物成像应用。基于它们的光物理性质，选择Mn4作进一步研究，因为它提供了最高的双光子活性和均匀的颗粒分布和形态。这四种化合物在24小时的孵育期内对细胞活性具有极低的细胞毒性作用，可用于生物研究。在活细胞中观察到强荧光信号，通过共聚焦荧光显微术对正常细胞和癌细胞进行固定细胞成像。这些结果证明，所制备的有机锰配合物可用于具有高信噪比的细胞成像。

目录

声明

Acknowledgements

摘要

ABSTRACT

Table of Contents

Chapter 1．Introduction of fluorescence bioimaging techniques and fluorescent probes

1．2．Fluorescence microscopy

1．2．1．Wide-field fluorescence microscopy(WFFM)

1．2．2．Confocal microscopy

1．2．3．Two-photon fluorescence microscopy

1．3．Fluorescence-lifetime imaging microscopy(FLIM)

1．3．1．FRET Imaging

1．3．2．Time-correlated single photon counting(TCSPC)

1．4．Super-resolution microscopy(SRM)

1．4．1．Stimulated emission depletion(STED)microscopy

1．5．Fluorescent bioimaging probes

1．5．1．Small organic two-photon fluorescence probe

1．5．2．Two-photon fluorescence probes for subcellular targeting

1．5．3．Two-photon fluorescent probes to detect intracellular metal ions

1．5．4．Two-photon fluorescent probes for Nucleic acids(NAs)staining

1．5．5．Detection of microenvironment in cells by two-photon fluorescence probes

1．5．6．Two-photon metal complexes

1．6．Aims and outlines of thesis

References

Chapter 2．A series of water-soluble pyridinium derivatives with two-photon absorption in the near infrared region for mitochondria targeting under stimulated emission depletion(STED)nanoscopy

2．1．Introduction

2．2．Experimental methods

2．2．2．Synthetic procedures of NL1-3 and PL1-3

2．2．3．Cell culture

2．2．4．Cytotoxieity and photostability of NL1-3 and PL1-3

2．2．5．Animal studies

2．2．7．Stimulated emission depletion(STED)nanoseopy

2．3．Results and discussions

2．3．1．Crystal structures，UV-Vis absorption and emission spectra，and TD-DFT studies of the target compounds

2．3．2．One-photon emission and two-photon absorption cross section response to solvent viscosity

2．3．3．Biological imaging applications of NL1

Refefences

Chapter 3．A series of two-photon absorption pyridinium sulfonate inner salts targeting endoplasmie reticulum(ER)，inducing cellular stress and mitochondria-mediated apoptosis in cancer cells

3．1．Introduction

3．2．Experimental

3．2．1．Materials and apparatus

3．2．2．Synthetic procedures of TriphenER1-2 and DiphenthioER1-2

3．2．3．Fluorescence-activated cell sorter(FACS)analysis of cellular uptake

3．2．4．Cell viability／proliferation Assay

3．2．5．Cell culture

3．2．6．Cell imaging using eonfoeal laser scanning microscopy

3．2．7．Stimulated emission depletion(STED)nanoseopy

3．2．8．Western Blot

3．3．Results and discussion

3．3．1．Crystal structures

3．3．2．Photophysical properties

3．3．3．Lipophilicity and cellular uptake of TriphenER1-2 and DiphenthioER1-2

3．3．4．Intracellular localization of DiphenthioER1 and Two-photon imaging application

3．3．5．DiphenthioER1 induced Endoplasmie Reticulum Stress and Nuclear Misshaping

3．3．6．Mitochondrial fragmentation and apoptosis

References

Chapter 4．DiphenthioER1；a two-photon active pyridinium derivative causing temporal opening of blood brain barriers via junctional proteins downregulation

4．1．IntrOduction

4．2．Experimental section

4．2．1．Cell culture and subculture

4．2．2．Cytotoxicity of DiphenthioER1 towards bEND.3 cells

4．2．4．3D in vitro BBB model set up

4．2．5．Preparation of trans-well slides for microscopy：Protocol

4．3．Results and discussions

4．3．2．Effect of DiphenthioER1 on tight junction(TJ) protein expression in 2D and 3D cultures

References

Chapter 5．SL-Neu；a two-photon fluorescent chemical probe for NeuN specific live neuron labeling

5．1．Introduction

5．2．Experimental

5．2．1．Reagents and instruments

5．2．2．Synthesis and characterization

5．2．3．Animal studies

5．2．3．Cell imaging using confocal laser scanning microscopy

5．2．4．Stimulated emission depletion(STED)nanoscopy

5．3．Results and discussions

5．3．1．Crystal structure and analysis

5．3．2．Linear optical properties

5．3．3．Nonlinear optical properties

5．3．4．Biological applications of SL-Neu

References

Chapter 6：Characterization and biological applications of terpyridine Mn2+ complexes

6．1．Introduction

6．2．Experimental section

6．2．1．Reagents and instruments

6．2．2．Synthesis of mononuclear Mn complexes

6．2．3．Determination of Size and morphology of Mn1-Mn4

6．2．4．Cytotoxieity by MTT assay

6．2．5．Cell culture

6．2．6．Cell imaging using confocal laser scanning microscopy

6．3．Results and discussions

6．3．1．Single crystal crystal structure and structure analysis

6．3．2．UV-vis absorption spectra of terpyridine-manganese complexes

6．3．2．One photon fluorescence spectroscopy

6．3．3．Fluorescence quantum yield and fluorescence lifetime

6．3．4．Two-photon induced fluorescence

6．3．5．Size and morphology determination by DLS，SEM and TEM

6．3．6．Cytotoxicity assay

6．3．7．Cellular imaging application of Mn4

References

Conclusions

Publications