| In situ real time, single particle tracking(SPT) technique has become a remarkable tool for the analysis of biological processes and organizational structure. Gold nanoparticles(Au NPs) are regarded as excellent probes for SPT, in addition to their unique optical properties, they are resistant to photobleaching and blinking, allowing them to be tracked for extended periods of time during important biological events. Many biological systems are inherently three-dimensional(3D) and biological processes usually occur in three dimension. Accordingly, 3D imaging technique is highly desired to detect in real time the accurate 3D-position variations of the particles in complex cellular processes to study the mechanisms of biomolecules behaviors and functions. Based on those, we choose Au NPs as probes, by using the darkfield optical microscope as an imaging system, a range of work was carried out as follows:In chapter 2, in order to improve the axial localization precision of the SPT technique, we developed a 3D darkfield imaging method of Au NPs, by inserting a long focal length cylindrical lens into the optical path of a darkfield microscope to break the axial symmetry of the optical system, which separates the axial focus of the two lateral dimensions and creates an elliptical point spread function(PSF) capable of describing the 3D position of the nanoparticle. We further applied this method in locating the position of Au NPs in pericellular matrix(PCM) and cell plasma membrane layer in the z direction. From the spatial-temporal distributions in the cell, we could exclude the thickness of the PCM layer was 5.35 μ m, which laid the foundation on studying the interaction of nanopartilces with PCM.In chapter 3, based on the 3D darkfield imaging method, we achieved a 3D super-resolution imaging with 28-36 nm laterally and 68 nm axially at a frame rate of 13 Hz. T he improved spatial precision made the approach very useful for 3D tracking, and enhanced time resolution allowed us to record the detailed time course of the cellular uptake process. With multiple resolutions both in 3D space and time of this approach, we were able to make the very direct measurement of the entire 3D trafficking itinerary of cell-penetrating peptide(CPP) modified Au NPs undergoing transmembrane process from the plasma membrane surface to eventually deep inside the cell, and revealed the details of the time course of the transmembrane process, which could help to study the hidden mechanistic steps underlying the programmed trafficking of CPP mediated delivery of nanocargos.In chapter 4, we present a darkfield system for orientational sensing with colorimetric analysis of single gold nanorod(Au NR). Anisotropic Au NR has been exploited for colorimetric polarization sensing, according to the color change of the scatterring light intensity of the Au NR in different polarization angles. By rotationg the polarizer in the optical path with changing the polarization of the linear polarized illumination relative to the orientation of the Au NR, it can be seen that the scattering color intensity of single Au NR change periodically and ignificantly follow a typical approximate cosine squared dependence on the angle between polarization direction and the Au NR long axis. T hen we further applied this system to study the rotational diffusion dynamics of Au NRs in glycerol solution, and we also demonstrated the application of the system to track the rotational dynamics of Au NRs in live cells. T his imaging technique holds great promise for polarization-controlled colorimetric nanomaterials and rotational motion in biomolecules. |
PDF Full Text Request