单分散金纳米棒的合成及其成像方法的研究应用
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摘要
生物学的研究中,已经从研究群体的共性转为寻找群体之中个体的差异性,进而研究个体的差异对整个群体的影响,单分子检测技术无疑是研究个体差异性的必要手段。它针对单个分子、单个细胞、单个细胞中某个生物功能分子展开研究,以期解开生物体内复杂而高度协调的生命机制。荧光染料在单分子检测中是非常有用的探针,然而它们不能在活细胞的单分子检测中提供足够的信号。开发新的探针用于检测单个活细胞内生物分子的作用机制,成为单分子技术发展的一个迫切需求。具有表面等离子共振的贵金属纳米材料自产生开始便受到极大的关注,大量文献报道了贵金属纳米材料作为探针和药物载体在生物学系统中的应用。伴随着贵金属纳米材料的研究进展,针对贵金属纳米粒子的单分子成像技术也迅速发展。本论文研究单个金纳米棒的成像新方法,主要从以下三个方面展开讨论:
(1)正交检偏显微镜对金纳米棒选择性成像研究
利用正交检偏光路,可以实现对各向异性纳米颗粒的选择性成像;高数值孔径的物镜,使成像时的景深小,纵向移动样品的位置,可以实现纵向切割成像,数据整合后达到三维成像的效果。两者相结合便可以实现只针对各向异性纳米材料的三维成像。金纳米棒是比较简单的各向异性纳米材料,而且其光性质稳定,可以在其表面修饰各种生物学配体,被用作探针来研究其在单个细胞内的活动。
(2)强散射金纳米棒的合成及其散射光与偏振光方向的关系
直径固定时,增加长径比,金纳米棒的消光横截面积并没有明显的增加。相同长径比的金纳米棒,直径越大,表面电子越多,表面等离子共振的效应越明显,散射信号越强。在晶种生长法合成的金纳米棒的基础上,继续让其生长,再生长过程中,不加入AgNO3,不以长轴生长方向为主,制备出直径约30～40nm,长约70nm的金纳米棒,用作单分子实验。初步研究了金纳米棒长轴与入射偏振光方向对散射光的影响,散射光强度与两者相对角度余弦值的平方成正比。暗场聚光镜改变了入射光的偏振方向,使入射光偏振方向不单一,在z轴上有分量。即使经过起偏器后的偏振光方向与金纳米棒长轴方向垂直,也不能够得到纯短轴的散射信号。即使入射光的偏振方向发生360°变化,金纳米棒颜色变化也呈现不出红绿逐渐变化的趋势。
(3)平面偏振光研究金纳米棒在细胞中的扩散
利用荧光偏振成像原理,将金纳米棒的成像过程分为激发和散射两个部分,激发和散射都与金纳米棒的空间角度相关。具体实验中利用平面偏振光激发样品,利用渥拉斯顿棱镜,将散射的信号分成两个相互垂直的偏振光。从收集到两个通道的点强度及其强度和,推导出金纳米棒的空间角度。同时研究了表面化学修饰物,溶液粘度对金纳米棒转动的影响。在单细胞内,金纳米棒呈现不同的运动轨迹,同时过程中伴随着金纳米棒自身的旋动运动。
Biology researches have shifted from studying common characteristics of groups, to individual differences among a group, then to how these differences affect the whole group. To study these differences, single molecule detection technique has gradually become a necessary means in routine experiments. This technique focuses on investigating behaviors of single molecules, single cells, or specific functional biomolecules in a single cell in order to resolve the complex and highly coordinated life mechanisms of organisms. In most single molecule detections, fluorescent dyes are very useful probes. However, in single molecule detection in living cells, the intensity of signals that dyes provide is not adequate to be detected. So it is greatly needed to develop new probes with potential application in detecting the mechanisms of biological molecules in single living cells. Noble metal nanomaterials with surface plasmon resonance have received great attentions since they were initially synthesized. There is a large pool of literature that reported the application of these nanomaterials as probes and drug carriers in biological systems. With the study progress of noble metal nanomaterial, single molecule imaging technique using noble metal nanoparticles has also developed quickly. Here, we study new imaging methods of single gold nanorods, which is mainly to be discussed in the following three aspects:
(1) Selective imaging gold nanorods using Polarization Microscopy
Selective imaging of anisotropic nanoparticles can be realized with polarizing microscopy and three dimensional scanning of sample using objective with high numerical aperture. With high NA value, the depth of field of the objective is thin, so the axial sectioning of sample is possible when moving objective vertically. Therefore, by coupling polarization microscope with an objective with high NA, it is capable to achieve three dimensional imaging of anisotropic nanoparticles. Gold nanorods are relatively simple anisotropic nanomaterial. Their optical properties are stable and they can be modified with a variety of biological ligands, so they are suitable probes to study activities in single cells.
(2) Synthesis of gold nanorods with intense scattering and light polarization dependence of scattering signals
For gold nanorods with a constant diameter, an increase in their aspect ratio would not result in any considerable increase of their extinction cross-section values. While for gold nanorods with the same aspect ratio, larger diameter would result in more surface electrons, stronger surface plasmon resonance effect, as well as stronger scattering signals. Gold nanorods, which are synthesized using seed mediated method, are allowed to overgrow without silver nitrate during the growth process so that their long axis would not become their major growth direction. In this way, we can obtain gold nanorods with diameter of about 30～40nm, and length of about 70nm. Is has been demonstrated that the scattering light of these anisotropic gold nanorods is strongly polarized along their long axis, making them in principle perfect orientation probes for single molecule experiment. The intensity of scatterring light is strongly dependent on the square of the cosine of the relative angle between the long axis and the polarization light. Dark field condenser changes the polarization direction of the incident light, making the polarization directions different. Therefore, we cannot obtain pure scattering signals along the short axis of gold nanorods even if the polarization direction is perpendicular to the long axis of gold nanorods. Also, the trend of gradual color change between read and green is not observed.
(3) Imaging of translational and rotational diffusion of gold nanorods in cells using planar illumination microscopy.
According to the similar theory of polarized fluorescence imaging, we can divide the imaging process of gold nanorods into two parts: absorption and scattering, which are both associated with the spatial angles of the gold nanorods. In detail, samples are illuminated with planar polarized light, and by passing through Wollaston prism, their scattering signals are divided into two polarized lights, which are mutually perpendicular. We can obtain the spatial angles of the gold nanorods by detecting the intensities of the signals of the polarized lights from the two channels. We also study the effect of surface modifications and the impact of the solution viscosity on the rotational dynamics of gold nanorods. We find that in a single cell, the trajectory of gold nanorods is different, accompanying with their own rotating movement.
(1)正交检偏显微镜对金纳米棒选择性成像研究
利用正交检偏光路,可以实现对各向异性纳米颗粒的选择性成像;高数值孔径的物镜,使成像时的景深小,纵向移动样品的位置,可以实现纵向切割成像,数据整合后达到三维成像的效果。两者相结合便可以实现只针对各向异性纳米材料的三维成像。金纳米棒是比较简单的各向异性纳米材料,而且其光性质稳定,可以在其表面修饰各种生物学配体,被用作探针来研究其在单个细胞内的活动。
(2)强散射金纳米棒的合成及其散射光与偏振光方向的关系
直径固定时,增加长径比,金纳米棒的消光横截面积并没有明显的增加。相同长径比的金纳米棒,直径越大,表面电子越多,表面等离子共振的效应越明显,散射信号越强。在晶种生长法合成的金纳米棒的基础上,继续让其生长,再生长过程中,不加入AgNO3,不以长轴生长方向为主,制备出直径约30～40nm,长约70nm的金纳米棒,用作单分子实验。初步研究了金纳米棒长轴与入射偏振光方向对散射光的影响,散射光强度与两者相对角度余弦值的平方成正比。暗场聚光镜改变了入射光的偏振方向,使入射光偏振方向不单一,在z轴上有分量。即使经过起偏器后的偏振光方向与金纳米棒长轴方向垂直,也不能够得到纯短轴的散射信号。即使入射光的偏振方向发生360°变化,金纳米棒颜色变化也呈现不出红绿逐渐变化的趋势。
(3)平面偏振光研究金纳米棒在细胞中的扩散
利用荧光偏振成像原理,将金纳米棒的成像过程分为激发和散射两个部分,激发和散射都与金纳米棒的空间角度相关。具体实验中利用平面偏振光激发样品,利用渥拉斯顿棱镜,将散射的信号分成两个相互垂直的偏振光。从收集到两个通道的点强度及其强度和,推导出金纳米棒的空间角度。同时研究了表面化学修饰物,溶液粘度对金纳米棒转动的影响。在单细胞内,金纳米棒呈现不同的运动轨迹,同时过程中伴随着金纳米棒自身的旋动运动。
Biology researches have shifted from studying common characteristics of groups, to individual differences among a group, then to how these differences affect the whole group. To study these differences, single molecule detection technique has gradually become a necessary means in routine experiments. This technique focuses on investigating behaviors of single molecules, single cells, or specific functional biomolecules in a single cell in order to resolve the complex and highly coordinated life mechanisms of organisms. In most single molecule detections, fluorescent dyes are very useful probes. However, in single molecule detection in living cells, the intensity of signals that dyes provide is not adequate to be detected. So it is greatly needed to develop new probes with potential application in detecting the mechanisms of biological molecules in single living cells. Noble metal nanomaterials with surface plasmon resonance have received great attentions since they were initially synthesized. There is a large pool of literature that reported the application of these nanomaterials as probes and drug carriers in biological systems. With the study progress of noble metal nanomaterial, single molecule imaging technique using noble metal nanoparticles has also developed quickly. Here, we study new imaging methods of single gold nanorods, which is mainly to be discussed in the following three aspects:
(1) Selective imaging gold nanorods using Polarization Microscopy
Selective imaging of anisotropic nanoparticles can be realized with polarizing microscopy and three dimensional scanning of sample using objective with high numerical aperture. With high NA value, the depth of field of the objective is thin, so the axial sectioning of sample is possible when moving objective vertically. Therefore, by coupling polarization microscope with an objective with high NA, it is capable to achieve three dimensional imaging of anisotropic nanoparticles. Gold nanorods are relatively simple anisotropic nanomaterial. Their optical properties are stable and they can be modified with a variety of biological ligands, so they are suitable probes to study activities in single cells.
(2) Synthesis of gold nanorods with intense scattering and light polarization dependence of scattering signals
For gold nanorods with a constant diameter, an increase in their aspect ratio would not result in any considerable increase of their extinction cross-section values. While for gold nanorods with the same aspect ratio, larger diameter would result in more surface electrons, stronger surface plasmon resonance effect, as well as stronger scattering signals. Gold nanorods, which are synthesized using seed mediated method, are allowed to overgrow without silver nitrate during the growth process so that their long axis would not become their major growth direction. In this way, we can obtain gold nanorods with diameter of about 30～40nm, and length of about 70nm. Is has been demonstrated that the scattering light of these anisotropic gold nanorods is strongly polarized along their long axis, making them in principle perfect orientation probes for single molecule experiment. The intensity of scatterring light is strongly dependent on the square of the cosine of the relative angle between the long axis and the polarization light. Dark field condenser changes the polarization direction of the incident light, making the polarization directions different. Therefore, we cannot obtain pure scattering signals along the short axis of gold nanorods even if the polarization direction is perpendicular to the long axis of gold nanorods. Also, the trend of gradual color change between read and green is not observed.
(3) Imaging of translational and rotational diffusion of gold nanorods in cells using planar illumination microscopy.
According to the similar theory of polarized fluorescence imaging, we can divide the imaging process of gold nanorods into two parts: absorption and scattering, which are both associated with the spatial angles of the gold nanorods. In detail, samples are illuminated with planar polarized light, and by passing through Wollaston prism, their scattering signals are divided into two polarized lights, which are mutually perpendicular. We can obtain the spatial angles of the gold nanorods by detecting the intensities of the signals of the polarized lights from the two channels. We also study the effect of surface modifications and the impact of the solution viscosity on the rotational dynamics of gold nanorods. We find that in a single cell, the trajectory of gold nanorods is different, accompanying with their own rotating movement.
引文
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