基于纳米粒子探针的光学成像新方法研究
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摘要
生命科学的迅速发展要求人们从单细胞和单分子水平上原位、活体、实时地了解物质之间的相互作用以及生命的过程。近年来新兴的单分子光学成像技术以其高的灵敏度和分辨率正好适应这一发展的要求,而且它们自诞生以来就一直处于迅速的发展中,成为当今生命科学及相关学科研究的前沿和热点。与此同时,这些基于光学手段的生物成像技术除了光学构型的革新和改进之外在很大程度上还依赖于灵敏而稳定的光学探针的使用。理想的光学探针应该具有以下特点:信号强;稳定,不易发生光漂白;体积小,对标记的宿主分子影响小;能够与宿主分子可控结合;化学惰性。遗憾的是,至今没有任何一种光学探针达到理想探针的要求。
本论文主要从光学探针和光学成像方法两方面入手,通过选择并优化合适的纳米粒子以及改进现有的光学成像技术,发展基于纳米粒子探针的光学成像新方法以实现高灵敏、稳定、快速的生物成像。主要研究工作包括如下几个方面:
1)利用全内反射荧光成像技术分别对水相合成的CdTe量子点和有机相合成的CdSe/ZnS量子点进行了荧光成像,并且通过对其荧光强度的跟踪获得了量子点在单粒子尺度上的荧光发射轨迹。我们发现在巯基丙酸、谷胱甘肽等含巯基配体的水溶液中合成的CdTe量子点不存在有机相合成量子点的荧光闪烁现象。进一步的实验证实巯基配体对量子点闪烁的抑制起到了至关重要的作用。最后,我们使用水相合成的CdTe量子点作为荧光探针成功地用于癌细胞的靶向成像。这些研究结果表明水相合成的CdTe量子点由于具有良好的水溶性,高亮度及不闪烁的特征可作为较好的荧光探针用于单分子检测及生物成像领域。
2)分别使用系综荧光光谱、荧光相关光谱、荧光显微镜等不同的光学技术研究了粒径范围在16 ~ 55 nm的金纳米粒子的光学性质,发现该粒径范围的金纳米粒子具有一定的荧光。而且,随着粒径的增加,金纳米粒子的荧光发射波长几乎保持不变,约为610 nm,其发射峰的半峰宽约为17 nm。尽管这些金纳米粒子的荧光量子产率很低,但在较高激发强度下它们具有足够强的荧光亮度可在单粒子尺度上被荧光显微镜和荧光相关光谱仪器所检测。尤其重要的是,在强光照射下,金纳米粒子不漂白。基于金纳米粒子的抗漂白性质以及细胞自荧光易漂白的特点,我们发展了一种以金纳米粒子作为荧光探针的细胞成像新方法。该方法的主要原理是活细胞经金纳米粒子培育或特异性标记后,利用光照将细胞自荧光迅速漂白,然后观察细胞内部或细胞膜上的金纳米粒子。基于上述原理,我们使用金纳米粒子作为探针或者靶向探针成功实现HeLa癌细胞的荧光成像。而且,利用全内反射荧光显微镜甚至不需要经过光漂白步骤就能够实现以金纳米粒子作为荧光探针的细胞膜成像。
3)金纳米粒子在光照下由于表面等离子体共振效应会产生强烈的散射信号,然而目前使用金纳米粒子作为散射光探针的生物成像方法主要局限于暗场显微镜、视频增强微分干涉相衬显微镜等传统光学成像技术。本文基于全内反射荧光显微镜平台,设计了一套物镜型隐失波散射成像系统。该系统通过使用一系列自制毫米级小孔对金纳米粒子的散射光和激光的反射光束进行有效分离,实现单个金纳米粒子的隐失波散射检测及溶液中单个金纳米粒子的快速跟踪。在本章中,我们详细阐述了物镜型隐失波散射成像的实现原理以及研究了激光入射角、小孔孔径和视场光阑开口直径等参数对该系统散射成像的影响。结果表明:通过适当地调节激光光束的入射角,小孔可应用的孔径范围位于2.5 mm到4 mm之间。最后,我们使用该隐失波散射成像技术成功实现了活细胞膜上单个金纳米粒子的跟踪,并且进一步研究了单个金纳米粒子在活细胞膜上的扩散行为并计算其相应的扩散系数。我们的研究结果表明以金纳米粒子作为光学探针的隐失波散射成像技术是一种非常具有前途的用以研究活细胞膜动力学的工具。而且,物镜型的光学配置构型更便于样品的放置,使得这种以金纳米粒子作为散射光探针的表面成像方法与膜片钳、原子力显微镜、扫描电化学显微镜等其他探测技术联用成为可能。
Single molecule optical imaging techniques provide great potential for understanding biological processes at the molecular level and for sensitive cancer diagnosis. From the day of their birth, single molecule optical imaging techniques have been the important research frontier and hotspots of life science and related disciplines. Besides the innovation of optical imaging principles and configurations, biological imaging with optical technique also relies greatly upon the use of sensitive and stable optical probes. An ideal optical probe for macro-molecules should generate an intense optical signal; it should also be small, durable, chemically inert, and apt to bind to the molecule of interest in a controlled manner. All currently-used optical markers fall short of the“ideal probe”status.
In this dissertation, combining nanoparticle technique with optical imaging methods, we develop new optical imaging methods for highly sensitive, stable and fast biological imaging. The main contributions are as follows:
1) We used total internal reflection fluorescence microscopy (TIRFM) to image clearly individual CdTe quantum dots (QDs) synthesized in aqueous solution and CdSe/ZnS QDs synthesized in organic phase, and investigated their fluorescence emission behavior under continous laser illumination.We found that individual CdTe QDs synthesized in mercaptopropionic acid (MPA) or glutathione (GSH) solution did not blink while CdSe/ZnS QDs synthesized in organic phase exhibited severe fluorescence blinking behavior. Our experiments have confirmed that the MPA and GSH coating on the CdTe QDs play a key role in suppressing blinking. Furthermore, we conjugated CdTe QDs to anti-epidermal growth factor receptor (anti-EGFR) antibodies, and successfully used the anti-EGFR/GNPs conjugates as targeted probes for fluorescent imaging of cancer cells. These results demonstrated that CdTe QDs synthesized in aqueous solution are well suitable for use in single molecule detection and biological imaging as fluorescent probes as they are water-soluble, biocompatible, bright, and non-blinking.
2) We investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behaviors of anti-photobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at single particle level. Based on this excellent anti-photobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. Based on this principle, we imaged living HeLa cells using GNPs as fluorescent probes, and obtained good cell images by photobleaching of cellular autofluorescence. In particular, under some specific illumination such as total internal reflection fluorescence microscopy, GNPs can also be directly used, not requiring the photobleaching procedure. Our preliminary results demonstrated that GNPs are good fluorescent probes in cell imaging and the cellular imaging method described has potential applications in cancer diagnostics and studies and immunoassays.
3) We propose a novel evanescent wave scattering imaging method using an objective-type total internal reflection system to image and track single gold nanoparticles (GNPs) in solution. In this imaging system, a millimeter-scale hole is only employed to efficiently separate GNPs scattering light from the background reflected beam. The detailed experimental realization of the imaging system was discussed, and the effect of the hole size on imaging was investigated. The experimental results showed that the hole diameters from 2.5 mm to 4 mm were suitable for the scattering imaging by adjusting the incidence angle. Furthermore, we applied the technology successfully to track single GNPs bound to live cell membrane via the anti-epidermal growth factor receptor antibody, and measured the diffusion coefficients of single particles by recording their corresponding trajectories. Compared to fluorescent dyes or quantum dots, GNPs have no photobleaching and no blinking, and the evanescent wave scattering imaging methods based on GNPs will become a very useful tool to study membrane dynamics in living cells. Additionally, the objective-based configuration provides a free space above the coverslip, and allows imaging and concomitant manipulation of live cells in culture by microinjection, patch-clamping, AFM and other techniques.
本论文主要从光学探针和光学成像方法两方面入手,通过选择并优化合适的纳米粒子以及改进现有的光学成像技术,发展基于纳米粒子探针的光学成像新方法以实现高灵敏、稳定、快速的生物成像。主要研究工作包括如下几个方面:
1)利用全内反射荧光成像技术分别对水相合成的CdTe量子点和有机相合成的CdSe/ZnS量子点进行了荧光成像,并且通过对其荧光强度的跟踪获得了量子点在单粒子尺度上的荧光发射轨迹。我们发现在巯基丙酸、谷胱甘肽等含巯基配体的水溶液中合成的CdTe量子点不存在有机相合成量子点的荧光闪烁现象。进一步的实验证实巯基配体对量子点闪烁的抑制起到了至关重要的作用。最后,我们使用水相合成的CdTe量子点作为荧光探针成功地用于癌细胞的靶向成像。这些研究结果表明水相合成的CdTe量子点由于具有良好的水溶性,高亮度及不闪烁的特征可作为较好的荧光探针用于单分子检测及生物成像领域。
2)分别使用系综荧光光谱、荧光相关光谱、荧光显微镜等不同的光学技术研究了粒径范围在16 ~ 55 nm的金纳米粒子的光学性质,发现该粒径范围的金纳米粒子具有一定的荧光。而且,随着粒径的增加,金纳米粒子的荧光发射波长几乎保持不变,约为610 nm,其发射峰的半峰宽约为17 nm。尽管这些金纳米粒子的荧光量子产率很低,但在较高激发强度下它们具有足够强的荧光亮度可在单粒子尺度上被荧光显微镜和荧光相关光谱仪器所检测。尤其重要的是,在强光照射下,金纳米粒子不漂白。基于金纳米粒子的抗漂白性质以及细胞自荧光易漂白的特点,我们发展了一种以金纳米粒子作为荧光探针的细胞成像新方法。该方法的主要原理是活细胞经金纳米粒子培育或特异性标记后,利用光照将细胞自荧光迅速漂白,然后观察细胞内部或细胞膜上的金纳米粒子。基于上述原理,我们使用金纳米粒子作为探针或者靶向探针成功实现HeLa癌细胞的荧光成像。而且,利用全内反射荧光显微镜甚至不需要经过光漂白步骤就能够实现以金纳米粒子作为荧光探针的细胞膜成像。
3)金纳米粒子在光照下由于表面等离子体共振效应会产生强烈的散射信号,然而目前使用金纳米粒子作为散射光探针的生物成像方法主要局限于暗场显微镜、视频增强微分干涉相衬显微镜等传统光学成像技术。本文基于全内反射荧光显微镜平台,设计了一套物镜型隐失波散射成像系统。该系统通过使用一系列自制毫米级小孔对金纳米粒子的散射光和激光的反射光束进行有效分离,实现单个金纳米粒子的隐失波散射检测及溶液中单个金纳米粒子的快速跟踪。在本章中,我们详细阐述了物镜型隐失波散射成像的实现原理以及研究了激光入射角、小孔孔径和视场光阑开口直径等参数对该系统散射成像的影响。结果表明:通过适当地调节激光光束的入射角,小孔可应用的孔径范围位于2.5 mm到4 mm之间。最后,我们使用该隐失波散射成像技术成功实现了活细胞膜上单个金纳米粒子的跟踪,并且进一步研究了单个金纳米粒子在活细胞膜上的扩散行为并计算其相应的扩散系数。我们的研究结果表明以金纳米粒子作为光学探针的隐失波散射成像技术是一种非常具有前途的用以研究活细胞膜动力学的工具。而且,物镜型的光学配置构型更便于样品的放置,使得这种以金纳米粒子作为散射光探针的表面成像方法与膜片钳、原子力显微镜、扫描电化学显微镜等其他探测技术联用成为可能。
Single molecule optical imaging techniques provide great potential for understanding biological processes at the molecular level and for sensitive cancer diagnosis. From the day of their birth, single molecule optical imaging techniques have been the important research frontier and hotspots of life science and related disciplines. Besides the innovation of optical imaging principles and configurations, biological imaging with optical technique also relies greatly upon the use of sensitive and stable optical probes. An ideal optical probe for macro-molecules should generate an intense optical signal; it should also be small, durable, chemically inert, and apt to bind to the molecule of interest in a controlled manner. All currently-used optical markers fall short of the“ideal probe”status.
In this dissertation, combining nanoparticle technique with optical imaging methods, we develop new optical imaging methods for highly sensitive, stable and fast biological imaging. The main contributions are as follows:
1) We used total internal reflection fluorescence microscopy (TIRFM) to image clearly individual CdTe quantum dots (QDs) synthesized in aqueous solution and CdSe/ZnS QDs synthesized in organic phase, and investigated their fluorescence emission behavior under continous laser illumination.We found that individual CdTe QDs synthesized in mercaptopropionic acid (MPA) or glutathione (GSH) solution did not blink while CdSe/ZnS QDs synthesized in organic phase exhibited severe fluorescence blinking behavior. Our experiments have confirmed that the MPA and GSH coating on the CdTe QDs play a key role in suppressing blinking. Furthermore, we conjugated CdTe QDs to anti-epidermal growth factor receptor (anti-EGFR) antibodies, and successfully used the anti-EGFR/GNPs conjugates as targeted probes for fluorescent imaging of cancer cells. These results demonstrated that CdTe QDs synthesized in aqueous solution are well suitable for use in single molecule detection and biological imaging as fluorescent probes as they are water-soluble, biocompatible, bright, and non-blinking.
2) We investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behaviors of anti-photobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at single particle level. Based on this excellent anti-photobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. Based on this principle, we imaged living HeLa cells using GNPs as fluorescent probes, and obtained good cell images by photobleaching of cellular autofluorescence. In particular, under some specific illumination such as total internal reflection fluorescence microscopy, GNPs can also be directly used, not requiring the photobleaching procedure. Our preliminary results demonstrated that GNPs are good fluorescent probes in cell imaging and the cellular imaging method described has potential applications in cancer diagnostics and studies and immunoassays.
3) We propose a novel evanescent wave scattering imaging method using an objective-type total internal reflection system to image and track single gold nanoparticles (GNPs) in solution. In this imaging system, a millimeter-scale hole is only employed to efficiently separate GNPs scattering light from the background reflected beam. The detailed experimental realization of the imaging system was discussed, and the effect of the hole size on imaging was investigated. The experimental results showed that the hole diameters from 2.5 mm to 4 mm were suitable for the scattering imaging by adjusting the incidence angle. Furthermore, we applied the technology successfully to track single GNPs bound to live cell membrane via the anti-epidermal growth factor receptor antibody, and measured the diffusion coefficients of single particles by recording their corresponding trajectories. Compared to fluorescent dyes or quantum dots, GNPs have no photobleaching and no blinking, and the evanescent wave scattering imaging methods based on GNPs will become a very useful tool to study membrane dynamics in living cells. Additionally, the objective-based configuration provides a free space above the coverslip, and allows imaging and concomitant manipulation of live cells in culture by microinjection, patch-clamping, AFM and other techniques.
引文
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