| Dark field microscopic imaging technology, as a non-scanning imaging technology with a high contrast, is widely used in a variety of fields, such as biochemical analysis, tracing of biological process and reaction monitoring. Single plasmonic nanoprobes have some advantages, such as the stable and strong optical signals, accurate localized information and the easily adjustable proterties, which are not available for the traditional probes, enabling the scattering imaging a wide application in reaction monitoring and spatial resolution at nanometer scale. Scattering enhancement induced by the plasmonic coupling is applied in development of nanoscale staff and biomedical analysis.However, there have been rare reports about reaction monitoring and research based on the scattering imaging technology nowadays and it may be due to the shortage of the suitable study model and the common correlation between the imaging signal and the dynamic reaction. In addition, the dark field microscopic imaging technology also has some shortages, for instance the imaging visibility is limited by the low use efficiency of the light source and spatial resolution is blocked by the optical diffraction limit.We carried out researches related to the dynamic reaction monitoring with scattering imaging and promotion of the imaging performance, and the main points of our work in this thesis are summarized as follows:1. Dark field microscopic imaging for the research of acid-sensitivity of the coordination polymers used as nano-sized drug carrier. By combining the organic ligand 1, 1’-(1,4-butanediyl) bis (imidazole) and the ferrous ions, coordination polymer spheres are prepared and used for effectively in situ encapsulating anticancer drug, doxorubicin hydrochloride (DOX·HC1). Through the regulation of the solvents, a high drug loading efficiency up to 98% and a drug loading content of nearly 40% can be obtained. This coordination polymer nanocarrier is pH-sensitive and with the conjugation of folic acid, it is used for the targeted drug delivery and the pH-sensitive controlled and sustained release. The targeted delivery can be confirmed by the fluorescence imaging, and the morphology of the microspheres in PBS at different time can be measured by SEM, but cannot be real-time monitored. However, the scattering imaging is successfully used for in situ real-time monitoring the pH-sensitive physical destabilization of the coordination polymer spheres. The destabilization rate difference at different pH is intuitional.2. Single nanoparticle scattering imaging for real-time monitoring of the light-induced chemical bond breakage. The photosensitivity of the covalently linked silver-dithiocarbamate (Ag-DTC) bond is real-time monitored by using both the spherical and rod shaped silver nanoprobes. The degradation of the DTC followed by the bond breakage leads to the formation of HS- in neutral condition. The formed HS-can induced the formation of an Ag2S shell on the surface of the silver nanoprobes, leading to a redshift of the LSPR scattering signal. The bond breakage can be effectively accelerated by the light irradiation, and some other influence factors, such as the pH, solvent polarity and reducing agents are studied comprehensively. The imaging date analyzed by software can confirm that the scattering intensity of the spherical nanoprobes firstly increase and then decrease until disappear, which is also confirmed by the single nanoparticle scattering spectra. The light-driven bond breakage is explained by the light-induced hot electrons coupling energy to the bond and the high redox active of silver, so this photosensitivity is not existing in the case of gold.3. Optical refit for visibility enhancement based on the light contribution induced by the surface plasmon. Usually, the adopted illumination method in dark field microscopic imaging is oblique illumination, through which the spatial separation of illumination and detection region and a high contrast is obtained. However, the low light utilization efficiency of this method leads to a poor visibility of the objects with weak scattering intensity, and thus it has to require high-intensity light source. The plasmonic nanoparticles, which have extinction cross sections larger than their physical cross sections, can contribute light into the adjacent region and scatter strong light. In the optical axis of the dry-type condenser U-DCD, a set of neutral density attenuators and filters is introduced to convert a part of the blocked light in dark field microscopic imaging into a color and intensity controlled monochromatic incident light, increasing the visibility of the plasmonic nanoparticles with the LSPR in this wavelength range. Both the imaging of 52 nm AgNPs and the AuNRs with a size of 75.4 nm in length and 38.5 nm in diameter obtain a larger scattering increase than the background and a well contrast is kept. The results are in consistent with the date analyzed with the software.4. Improved imaging technique applied for small nanoparticles and nanoparticles in cell imaging. In some biological analysis system, only the small sized nanoparticles could be used because the relatively low levels of steric hindrance was important. The scattering intensity of the plasmonic nanoparticles is in consistent with the six power of the radius of the particles, so the small particles have weak scattering intensity, restricting their wide applications. With the enhanced scattering imaging technology, both curcumin-coated 20 nm AgNPs and AuNRs with a size of 62.6 nm in length and 27.9 nm in diameter have greatly improved scattering imaging visibility, which can also be seen from the RGB line distribution analysis. Scattering imaging of complex samples are susceptible to interference from the background, thus the visibility of the probes are greatly reduced. In addition, dispersed 30 nm blue silver nanoparticles and the red aggregates of these AgNPs in Hep-2 cancer cells obtain high-visibility imaging.5. Influence of heterogeneous plasmonic optical imaging element on spatial resolution of dark field microscopic imaging. Since the establishment of nanoscale staff based on the coupling of plasmonic nanoparticles, it has been widely used in distance-related biochemical analysis. Due to the limited optical imaging resolution, the spots merge when the plasmonic nanoprobes coupled, so it’s hard to determine the precise location of the single particles from the optical imaging. Colocalization analysis of the coupling blue AgNP and red AuNR under the DFM and SEM shows that the blue color of AgNP and the red color of AuNR can both be seen in the optical images, which is absolute different from the coupling between the same nanoparticles. This heterogeneous plasmonic optical probes can be used for the better resolution of the monomer in coupling particles. DFM with a narrow light source and monochromatic imaging and information reconstruction technology can to some extent enhance the ability to distinguish the monomer in red and blue coupling nanoparticles.In summary, real-time reaction monitoring and the research of the related properties at nanometer scale is achieved by dark field microscopic imaging technology. In addition, the visibility of the scattering imaging of plasmonic nanoparticles has been increased obviously with the refit scattering imaging method, at the same time, the study model of direct resolution of the monomer in two close coupled particles from the scattering imaging has also been set up. From these design, more information could be obtained from the scattering imaging of the plasmonic nanoprobes. |
