Studies On Optical Imaging Methods Using Nanoparticles As Probes

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.