Ph.D. Defense: Han Zhang

    Monday, November 24, 2014 - 9:00am
    Meinel 447

    "Superresolution Nonlinear Structured Illumination Microscopy by Stimulated Emission Depletion"


    The understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy has been demonstrated a fast and full-field superresolution imaging tool, with applications such as Saturated-SIM and Photoswitching-SIM.

    In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretically unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full-field imaging over a 10×10 micron square at the speed of two seconds per frame with four-fold improved resolution. Our STED-SIM technique has been applied on biological samples, and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. 

    In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance, to nonlinear STED-SIM has been made and around eight-time enhancement on STED quenching effect was achieved. And the feasibility of 3-D STED-SIM is also studied by simulation. The preliminary result shows the potential of achieving axial and lateral superresolution by 3-D STED-SIM.

    The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two-photon Bessel beam light-sheet microscopy and nonlinear SIM, 3-D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebra fish. Two-color imaging of pronephric glomeruli and vasculature of zebra fish kidney, whose cellular structures located at the center of the fish body, are revealed in high clarity.