【摘要】：暗場散射成像技術,作為一種高對比度和非掃描的光學成像技術,被廣泛的應用于分析傳感、生物過程示蹤以及反應監(jiān)控等領域。單納米顆粒等離子體探針,具有傳統(tǒng)散射探針無法比擬的優(yōu)勢,如穩(wěn)定且強的光學信號,精準的局域信息以及易于調控的性質,使得散射成像在納米尺度的動態(tài)反應監(jiān)控和空間分辨方面具有廣闊的應用前景。等離子體耦合引起的散射增強和光譜移動也被廣泛應用于納米標尺的開發(fā)和生物醫(yī)學分析檢測。然而,目前利用暗場散射成像來監(jiān)控和研究反應過程的報道為數(shù)不多,對于刺激響應型的可控反應的監(jiān)控更是鮮有報道,可能是由于缺乏適當?shù)难芯磕Ｐ鸵约俺上裥盘柡蛣討B(tài)反應難以建立有效的銜接。目前,暗場成像技術自身仍存在一定的局限性,包括可見度在一定程度上受限于較低的光源利用率,以及衍射極限的束縛使得的空間分辨受限。本論文圍繞上述的研究難點和問題,借鑒前人的工作基礎,在散射成像監(jiān)控動態(tài)反應過程以及成像性能改進方面開展了如下具體的工作：1.暗場散射成像對配合物納米藥物載體的酸敏感性的研究。利用有機配體1,1’-(1,4-丁基)二咪唑和亞鐵離子制備了配位聚合物微球,同時實現(xiàn)了對抗癌藥物鹽酸阿霉素的高效原位包覆。通過反應溶劑的調控,可以獲得最優(yōu)的高達98%的藥物負載率和近40%的藥物負載效率。此納米藥物載體由于其自身具有的pH敏感性,以及經(jīng)表面硅烷化后進行的癌細胞靶向劑葉酸的修飾,被用于抗癌藥物的靶向傳輸和pH敏感的緩釋和控釋。熒光成像結果表明此載體具有靶向作用,SEM對各時間點的微球形貌研究結果可以證明其酸敏感性,卻無法對單顆粒水平進行連續(xù)實時監(jiān)控,而暗場散射成像則實現(xiàn)了對酸敏感的微球載體降解過程的單顆粒水平的實時原位監(jiān)控,直觀地考察了藥物載體的酸敏感性。2.單顆粒散射成像對光致化學鍵斷裂反應的實時監(jiān)控。將球形和棒狀兩種形體的銀納米顆粒作為暗場成像下的檢測探針,實現(xiàn)了對共價連接的銀-二硫代氨基甲酸(Ag-DTC)化學鍵的光敏感性的實時監(jiān)控。通過觀察Ag-DTC斷裂后所伴隨產(chǎn)生的硫氫根介導形成的Ag@Ag2S核殼結構帶來的散射信號紅移,發(fā)現(xiàn)此化學鍵的斷裂可以有效地被光驅動,同時對另外幾個重要的影響因素,比如pH,溶劑極性以及還原劑等,也進行了全面的考察。借助軟件進行成像數(shù)據(jù)分析,可以精確地發(fā)現(xiàn)球形AgNPs探針的散射強度在反應監(jiān)控過程中,存在一個先升高后降低的過程,單顆粒散射光譜也證實了這一現(xiàn)象。對于光的驅動機制解釋為光生熱電子克服Ag-DTC斷鍵的能壘,同時結合銀的較高的氧化還原活性,從而表現(xiàn)出Au-DTC所不具有的光敏感性。3.基于表面等離激元“光濃縮”效應和光路改裝提升成像可見度的研究。目前,暗場成像主要采用斜射照明,通過入射光與樣品的散射光的空間分離獲得優(yōu)異的成像對比度。如此一來,光源的利用效率比較低,從而使得弱散射信號的可見度受損,同時對光源的功率要求較高。等離子體納米顆粒具有大于物理截面的散射截面,從而可以將光集中到顆粒附近區(qū)域,從而散射出很強的光。在干式暗場聚光鏡U-DCD的光軸上添加一組濾光片和中性密度衰減片,將暗場成像中部分未利用的光源轉化為顏色和強度可調的單色入射光,用于增強具LSPR波長位于該顏色波段范圍內的等離子體顆粒的成像可見度,包括52 nm的AgNPs和長75.4 nm和直徑38.5 nm的AuNRs,獲得了強于引入的背景光的散射增強,同時保留了較好的成像對比度。使用軟件的分析結果與成像效果相符。4.改進的成像方法用于小粒徑顆粒和細胞中顆粒的散射成像。很多情況下,研究對象的尺寸和空間位阻的問題使得納米探針的選擇僅僅限于小粒徑的探針。等離子體納米顆粒的散射能力與半徑的6次方成正比,因此小粒徑的等離子體顆粒的散射強度較弱,這成為制約小粒徑探針廣泛使用的一個因素。借助上一章設計的增強散射成像可見度的設計,姜黃素包被的20 nm的球形AgNPs以及長62.6 nm,直徑27.9 nm的AuNPs成像可見度得到了明顯的提升,從成像和RGB線分布數(shù)據(jù)都可以得出以上結果。復雜樣品的散射成像容易受到背景的干擾,從而大幅度降低探針的可見度。同樣借助上述的方法,實現(xiàn)了對喉癌上皮細胞中的分散的30 nm藍色銀納米顆粒和紅色聚集體的高可見度成像。5.雙色等離子體光學探針對暗場成像分辨影響的研究�；诘入x子體納米顆粒耦合的納米標尺的方法自從被建立以來就在距離相關的生化分析得到廣泛應用。受限于光學成像分辨率,近距離的等離子體探針成像時容易出現(xiàn)光斑的歸并,從而難以從光學成像上對單體納米探針的位置進行精準的確定。對具有不同等離子體散射波段的兩個納米探針進行SEM和暗場散射成像的共定位分析,可以發(fā)現(xiàn),近距離耦合的藍色AgNP與紅色AgNR在暗場成像光斑中同時保留了紅色和藍色,這與相同形體的納米顆粒散射成像結果是截然不同的。借助這種可以保留各自光學信息的雙色等離子體納米探針可以用于光學上成像單體的更佳的分辨。借助窄光源的暗場成像和單色成像重構方法可以在一定程度上進一步提升紅藍顆粒的分辨能力�？傊�,本論文借助暗場散射成像技術實現(xiàn)了對納米尺度的反應實時監(jiān)控以及相關性質的考察和確定。另外通過改進的散射成像方法實現(xiàn)了對散射成像可見度的明顯提升,同時設計了通過散射成像上分辨雙顆粒中單體顆粒位置信息的研究模型,將有利于從等離子體納米探針的散射成像獲取更多的信息。
[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.
【學位授予單位】：西南大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：O439

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