暗场散射成像在刺激响应型反应监控中的应用及高性能成像系统研究
发布时间：2018-07-15 14:34
【摘要】：暗场散射成像技术,作为一种高对比度和非扫描的光学成像技术,被广泛的应用于分析传感、生物过程示踪以及反应监控等领域。单纳米颗粒等离子体探针,具有传统散射探针无法比拟的优势,如稳定且强的光学信号,精准的局域信息以及易于调控的性质,使得散射成像在纳米尺度的动态反应监控和空间分辨方面具有广阔的应用前景。等离子体耦合引起的散射增强和光谱移动也被广泛应用于纳米标尺的开发和生物医学分析检测。然而,目前利用暗场散射成像来监控和研究反应过程的报道为数不多,对于刺激响应型的可控反应的监控更是鲜有报道,可能是由于缺乏适当的研究模型以及成像信号和动态反应难以建立有效的衔接。目前,暗场成像技术自身仍存在一定的局限性,包括可见度在一定程度上受限于较低的光源利用率,以及衍射极限的束缚使得的空间分辨受限。本论文围绕上述的研究难点和问题,借鉴前人的工作基础,在散射成像监控动态反应过程以及成像性能改进方面开展了如下具体的工作：1.暗场散射成像对配合物纳米药物载体的酸敏感性的研究。利用有机配体1,1’-(1,4-丁基)二咪唑和亚铁离子制备了配位聚合物微球,同时实现了对抗癌药物盐酸阿霉素的高效原位包覆。通过反应溶剂的调控,可以获得最优的高达98%的药物负载率和近40%的药物负载效率。此纳米药物载体由于其自身具有的pH敏感性,以及经表面硅烷化后进行的癌细胞靶向剂叶酸的修饰,被用于抗癌药物的靶向传输和pH敏感的缓释和控释。荧光成像结果表明此载体具有靶向作用,SEM对各时间点的微球形貌研究结果可以证明其酸敏感性,却无法对单颗粒水平进行连续实时监控,而暗场散射成像则实现了对酸敏感的微球载体降解过程的单颗粒水平的实时原位监控,直观地考察了药物载体的酸敏感性。2.单颗粒散射成像对光致化学键断裂反应的实时监控。将球形和棒状两种形体的银纳米颗粒作为暗场成像下的检测探针,实现了对共价连接的银-二硫代氨基甲酸(Ag-DTC)化学键的光敏感性的实时监控。通过观察Ag-DTC断裂后所伴随产生的硫氢根介导形成的Ag@Ag2S核壳结构带来的散射信号红移,发现此化学键的断裂可以有效地被光驱动,同时对另外几个重要的影响因素,比如pH,溶剂极性以及还原剂等,也进行了全面的考察。借助软件进行成像数据分析,可以精确地发现球形AgNPs探针的散射强度在反应监控过程中,存在一个先升高后降低的过程,单颗粒散射光谱也证实了这一现象。对于光的驱动机制解释为光生热电子克服Ag-DTC断键的能垒,同时结合银的较高的氧化还原活性,从而表现出Au-DTC所不具有的光敏感性。3.基于表面等离激元“光浓缩”效应和光路改装提升成像可见度的研究。目前,暗场成像主要采用斜射照明,通过入射光与样品的散射光的空间分离获得优异的成像对比度。如此一来,光源的利用效率比较低,从而使得弱散射信号的可见度受损,同时对光源的功率要求较高。等离子体纳米颗粒具有大于物理截面的散射截面,从而可以将光集中到颗粒附近区域,从而散射出很强的光。在干式暗场聚光镜U-DCD的光轴上添加一组滤光片和中性密度衰减片,将暗场成像中部分未利用的光源转化为颜色和强度可调的单色入射光,用于增强具LSPR波长位于该颜色波段范围内的等离子体颗粒的成像可见度,包括52 nm的AgNPs和长75.4 nm和直径38.5 nm的AuNRs,获得了强于引入的背景光的散射增强,同时保留了较好的成像对比度。使用软件的分析结果与成像效果相符。4.改进的成像方法用于小粒径颗粒和细胞中颗粒的散射成像。很多情况下,研究对象的尺寸和空间位阻的问题使得纳米探针的选择仅仅限于小粒径的探针。等离子体纳米颗粒的散射能力与半径的6次方成正比,因此小粒径的等离子体颗粒的散射强度较弱,这成为制约小粒径探针广泛使用的一个因素。借助上一章设计的增强散射成像可见度的设计,姜黄素包被的20 nm的球形AgNPs以及长62.6 nm,直径27.9 nm的AuNPs成像可见度得到了明显的提升,从成像和RGB线分布数据都可以得出以上结果。复杂样品的散射成像容易受到背景的干扰,从而大幅度降低探针的可见度。同样借助上述的方法,实现了对喉癌上皮细胞中的分散的30 nm蓝色银纳米颗粒和红色聚集体的高可见度成像。5.双色等离子体光学探针对暗场成像分辨影响的研究。基于等离子体纳米颗粒耦合的纳米标尺的方法自从被建立以来就在距离相关的生化分析得到广泛应用。受限于光学成像分辨率,近距离的等离子体探针成像时容易出现光斑的归并,从而难以从光学成像上对单体纳米探针的位置进行精准的确定。对具有不同等离子体散射波段的两个纳米探针进行SEM和暗场散射成像的共定位分析,可以发现,近距离耦合的蓝色AgNP与红色AgNR在暗场成像光斑中同时保留了红色和蓝色,这与相同形体的纳米颗粒散射成像结果是截然不同的。借助这种可以保留各自光学信息的双色等离子体纳米探针可以用于光学上成像单体的更佳的分辨。借助窄光源的暗场成像和单色成像重构方法可以在一定程度上进一步提升红蓝颗粒的分辨能力。总之,本论文借助暗场散射成像技术实现了对纳米尺度的反应实时监控以及相关性质的考察和确定。另外通过改进的散射成像方法实现了对散射成像可见度的明显提升,同时设计了通过散射成像上分辨双颗粒中单体颗粒位置信息的研究模型,将有利于从等离子体纳米探针的散射成像获取更多的信息。
[Abstract]:Dark field scattering imaging, as a high contrast and non scanning optical imaging technology, is widely used in the fields of sensing, biological process tracer and reaction monitoring. Single nano particle plasma probe has the advantages of the traditional scattering probe, such as stable and strong optical signals and accurate local information. The scattering imaging has a wide application prospect in the field of dynamic response monitoring and spatial resolution in nanoscale. The scattering enhancement and spectrum movement caused by plasma coupling are also widely used in the development of nanoscale scale and biomedical analysis. There are few reports on the reaction process, and there are few reports on the controllable response monitoring of the stimulus response. It may be due to the lack of appropriate research models, imaging signals and dynamic responses that are difficult to establish effective cohesion. It is limited to low utilization of light source and restricted by the constraint of diffraction limit. This paper focuses on the research difficulties and problems mentioned above, drawing on the work basis of predecessors, carrying out specific work in the dynamic response process of the scattered imaging monitoring and the improvement of imaging performance: 1. the matching of the scattering imaging of the dark field Study on the acid sensitivity of nanoscale drug carriers. The coordination polymer microspheres were prepared by the organic ligand 1,1 '- (1,4- Ding Ji) two imidazole and ferrous ions, and the effective in situ encapsulation of adriamycin hydrochloride was realized. The optimal drug loading rate of up to 98% and nearly 40% of the drug could be obtained by the control of the reaction solvent. Load efficiency. The nanoscale drug carrier, due to its own pH sensitivity, and the modification of the cancer cell targeting agent folic acid after the surface silylation, is used for targeted transmission of anticancer drugs and pH sensitive release and controlled-release. The results of fluorescence imaging show that the carrier has a targeting effect and the microsphere morphology of SEM at various time points is studied. The results can prove its acid sensitivity, but can not continuously monitor the single particle level in real time, while the dark field scattering imaging realizes the real-time in situ monitoring of the single particle level of the acid sensitive microsphere carrier degradation process. The acid sensitivity.2. single particle scattering imaging of the drug carrier is directly investigated for the photochemical bond fracture reaction. Real time monitoring of the spherical and rod like silver nanoparticles as the detection probe under the dark field imaging, realized the real-time monitoring of the light sensitivity of the covalent connecting silver - two thiocarbamate (Ag-DTC) chemical bond. By observing the Ag@Ag2S nuclear shell formed by the thiohydrogen root induced by the Ag-DTC fracture The red shift of the scattered signal shows that the cleavage of the chemical bond can be effectively driven by light. At the same time, several other important factors, such as pH, solvent polarity and reducing agent, have also been thoroughly investigated. With the aid of the software analysis of imaging data, the scattering intensity of the spherical AgNPs probe can be accurately found in the process of monitoring the reaction. The single particle scattering spectrum also confirms this phenomenon. For the light driving mechanism, it is explained that photothermal electrons can overcome the energy barrier of the Ag-DTC broken bond and combine the high redox activity of silver, thus showing that the photosensitive.3. that Au-DTC does not have is based on the surface plasmon polariton "light concentration". The effect and the optical path modification is the research of the visibility of the lifting imaging. At present, the dark field imaging mainly uses the oblique illumination, and the excellent imaging contrast is obtained by separating the scattered light from the incident light. In this way, the use efficiency of the light source is low, thus the visibility of the weak scattering signal is damaged, and the power requirement of the light source is also required. The plasma nanoparticles have a scattering cross section that is larger than the physical section, so that the light can be concentrated in the vicinity of the particle, thus scattering a strong light. A group of filters and neutral density attenuation patches are added to the optical axis of the dry dark field spotlight U-DCD, and the light source in the dark field imaging is converted to color and strength. The adjustable monochromatic incident light is used to enhance the imaging visibility of plasma particles with LSPR wavelength in the range of the color band, including 52 nm AgNPs and 75.4 nm and AuNRs with diameter 38.5 nm. The enhanced scattering enhancement of the background light is obtained, and the better imaging contrast is retained. The analysis results and the results of the software are used. In many cases, the size and space hindrance of the object makes the selection of the nano probe limited to the small particle size probe. The scattering ability of the plasma nanoparticles is proportional to the 6 square of the radius, so the small size of the particle size can be obtained in many cases. The weak scattering intensity of plasma particles has become a factor restricting the wide use of small particle diameter probes. With the design of enhanced scattering imaging visibility designed in the last chapter, the visibility of the 20 nm spherical AgNPs of curcumin envelope and the AuNPs imaging of a diameter of 27.9 nm, with a diameter of 27.9 nm, has been significantly improved, from the imaging and RGB line distribution. The above results can be obtained by the data. The scattering imaging of complex samples is easily interfered by the background, thus greatly reducing the visibility of the probe. By the same method, the 30 nm blue silver nanoparticles and the high visibility imaging.5. dual color plasma optical exploration for the laryngeal carcinoma epithelial cells are realized. Research on the influence of dark field imaging resolution. The method of nano scale based on plasma nanoscale coupling has been widely used since it was established. Limited to the resolution of optical imaging, the near distance plasma probe imaging is easy to merge the spot of light, so it is difficult to image from optical imaging. The location of the nanoscale probe is accurately determined. A co localization analysis of the SEM and dark field scattering imaging of two nanoscale probes with different plasma scattering bands can be found. It is found that the near distance coupled blue AgNP and red AgNR retain both red and blue in the dark field imaging spot, which is the same as the same body. The results of scattering imaging of rice particles are completely different. With the use of this kind of dual color plasma nanoscale probe which can retain their respective optical information, the better resolution of optical imaging monomers can be used. The resolution of red and blue particles can be further improved by means of dark field imaging and monochromatic imaging reconstruction with narrow light source. In this paper, the real-time monitoring of the nanoscale reaction and the investigation and determination of the related properties are realized with the help of the dark field scattering imaging technology. In addition, the improved scattering imaging method has been used to improve the visibility of the scattering imaging. At the same time, the location information of the single particles in the double particles is resolved by the scattering imaging. The research model will help to get more information from the scattering imaging of plasma nanoprobes.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O439
[Abstract]:Dark field scattering imaging, as a high contrast and non scanning optical imaging technology, is widely used in the fields of sensing, biological process tracer and reaction monitoring. Single nano particle plasma probe has the advantages of the traditional scattering probe, such as stable and strong optical signals and accurate local information. The scattering imaging has a wide application prospect in the field of dynamic response monitoring and spatial resolution in nanoscale. The scattering enhancement and spectrum movement caused by plasma coupling are also widely used in the development of nanoscale scale and biomedical analysis. There are few reports on the reaction process, and there are few reports on the controllable response monitoring of the stimulus response. It may be due to the lack of appropriate research models, imaging signals and dynamic responses that are difficult to establish effective cohesion. It is limited to low utilization of light source and restricted by the constraint of diffraction limit. This paper focuses on the research difficulties and problems mentioned above, drawing on the work basis of predecessors, carrying out specific work in the dynamic response process of the scattered imaging monitoring and the improvement of imaging performance: 1. the matching of the scattering imaging of the dark field Study on the acid sensitivity of nanoscale drug carriers. The coordination polymer microspheres were prepared by the organic ligand 1,1 '- (1,4- Ding Ji) two imidazole and ferrous ions, and the effective in situ encapsulation of adriamycin hydrochloride was realized. The optimal drug loading rate of up to 98% and nearly 40% of the drug could be obtained by the control of the reaction solvent. Load efficiency. The nanoscale drug carrier, due to its own pH sensitivity, and the modification of the cancer cell targeting agent folic acid after the surface silylation, is used for targeted transmission of anticancer drugs and pH sensitive release and controlled-release. The results of fluorescence imaging show that the carrier has a targeting effect and the microsphere morphology of SEM at various time points is studied. The results can prove its acid sensitivity, but can not continuously monitor the single particle level in real time, while the dark field scattering imaging realizes the real-time in situ monitoring of the single particle level of the acid sensitive microsphere carrier degradation process. The acid sensitivity.2. single particle scattering imaging of the drug carrier is directly investigated for the photochemical bond fracture reaction. Real time monitoring of the spherical and rod like silver nanoparticles as the detection probe under the dark field imaging, realized the real-time monitoring of the light sensitivity of the covalent connecting silver - two thiocarbamate (Ag-DTC) chemical bond. By observing the Ag@Ag2S nuclear shell formed by the thiohydrogen root induced by the Ag-DTC fracture The red shift of the scattered signal shows that the cleavage of the chemical bond can be effectively driven by light. At the same time, several other important factors, such as pH, solvent polarity and reducing agent, have also been thoroughly investigated. With the aid of the software analysis of imaging data, the scattering intensity of the spherical AgNPs probe can be accurately found in the process of monitoring the reaction. The single particle scattering spectrum also confirms this phenomenon. For the light driving mechanism, it is explained that photothermal electrons can overcome the energy barrier of the Ag-DTC broken bond and combine the high redox activity of silver, thus showing that the photosensitive.3. that Au-DTC does not have is based on the surface plasmon polariton "light concentration". The effect and the optical path modification is the research of the visibility of the lifting imaging. At present, the dark field imaging mainly uses the oblique illumination, and the excellent imaging contrast is obtained by separating the scattered light from the incident light. In this way, the use efficiency of the light source is low, thus the visibility of the weak scattering signal is damaged, and the power requirement of the light source is also required. The plasma nanoparticles have a scattering cross section that is larger than the physical section, so that the light can be concentrated in the vicinity of the particle, thus scattering a strong light. A group of filters and neutral density attenuation patches are added to the optical axis of the dry dark field spotlight U-DCD, and the light source in the dark field imaging is converted to color and strength. The adjustable monochromatic incident light is used to enhance the imaging visibility of plasma particles with LSPR wavelength in the range of the color band, including 52 nm AgNPs and 75.4 nm and AuNRs with diameter 38.5 nm. The enhanced scattering enhancement of the background light is obtained, and the better imaging contrast is retained. The analysis results and the results of the software are used. In many cases, the size and space hindrance of the object makes the selection of the nano probe limited to the small particle size probe. The scattering ability of the plasma nanoparticles is proportional to the 6 square of the radius, so the small size of the particle size can be obtained in many cases. The weak scattering intensity of plasma particles has become a factor restricting the wide use of small particle diameter probes. With the design of enhanced scattering imaging visibility designed in the last chapter, the visibility of the 20 nm spherical AgNPs of curcumin envelope and the AuNPs imaging of a diameter of 27.9 nm, with a diameter of 27.9 nm, has been significantly improved, from the imaging and RGB line distribution. The above results can be obtained by the data. The scattering imaging of complex samples is easily interfered by the background, thus greatly reducing the visibility of the probe. By the same method, the 30 nm blue silver nanoparticles and the high visibility imaging.5. dual color plasma optical exploration for the laryngeal carcinoma epithelial cells are realized. Research on the influence of dark field imaging resolution. The method of nano scale based on plasma nanoscale coupling has been widely used since it was established. Limited to the resolution of optical imaging, the near distance plasma probe imaging is easy to merge the spot of light, so it is difficult to image from optical imaging. The location of the nanoscale probe is accurately determined. A co localization analysis of the SEM and dark field scattering imaging of two nanoscale probes with different plasma scattering bands can be found. It is found that the near distance coupled blue AgNP and red AgNR retain both red and blue in the dark field imaging spot, which is the same as the same body. The results of scattering imaging of rice particles are completely different. With the use of this kind of dual color plasma nanoscale probe which can retain their respective optical information, the better resolution of optical imaging monomers can be used. The resolution of red and blue particles can be further improved by means of dark field imaging and monochromatic imaging reconstruction with narrow light source. In this paper, the real-time monitoring of the nanoscale reaction and the investigation and determination of the related properties are realized with the help of the dark field scattering imaging technology. In addition, the improved scattering imaging method has been used to improve the visibility of the scattering imaging. At the same time, the location information of the single particles in the double particles is resolved by the scattering imaging. The research model will help to get more information from the scattering imaging of plasma nanoprobes.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O439
【共引文献】
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