UNIVERSITY OF MARYLAND

This application addresses broad the Challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-GM-106; subcellular imaging of metal ions. Methods to improve spatial resolution are complex and not always compatible with cell imaging. For example, near-field scanning optical microscopy (NSOM) requires near contact of the NSOM probe with the sample and can only be used to image the upper surface of cells. We propose to develop a novel type of microscopy technique that provides resolution several-fold better than diffraction limited optics and can be used to image metal ions in cells. It is known that subwavelength size electric field distributions can occur upon illumination of certain metallic nanostructures. We have now observed a unique phenomenon above metallic nanostructures which promises to provide sub-wavelength imaging resolution in all regions of the cell, not just the contact region. We have recently shown that sub-wavelength size fields can be created at substantial distances above the metallic structures, ranging from 1 to 10 microns. We believe this phenomenon can be used to excite sub-wavelength size volumes in cells and can provide the basis for a new class of optical microscopes. We propose to use this remarkable optical phenomenon to develop a novel microscope for imaging of metal ions in cells with a spatial resolution approaching 50 nm. The usefulness of this approach to imaging metal ions will be extended to many metal ions of interest by using fluorescence lifetime imaging microscopy (FLIM), which will also make the imaging to be less affected by photobleaching. We will use the finite-difference time-domain method to simulate the field distributions above metallic nanostructures (MNS). Since these structures will be used for intracellular ion imaging we will extend the calculations to 10 microns or more above the surface. The geometry of the MNS will be varied to obtain sub-wavelength volumes for the electric field. We will model nanohole arrays which will provide a patterned illumination and concentric nanorings which provide point illumination. Depending upon the intended wavelength range the structures will be made out of gold, silver or aluminum, which will extend the wavelength range from the UV to the NIR. We will use the focused ion bean (FIB) method to fabricate the initial structures. As needed we will use electron beam (EB) lithography to fabricate a larger number of progressively varying structures. EB is faster than FIB for larger patterns. We will prepare similar patterns in non-plasmonic materials like chromium to compare with the metal structure. We will use the requested NSOM instrument to measure the electric field distributions above the metallic structures. The NSOM results will then be used to refine the geometries of the MNS to obtain the highest spatial resolution. The individual spots will be 1 or more microns apart to allow for readout using far-field optics. The selected metal nanostructures will then be used with fluorescence and NSOM to determine the sizes of the excited volumes. These volumes will also be estimated using fluorescence correlation spectroscopy (FCS). The known metal ion concentrations in the solution will be compared with the concentrations determined from the wavelength-ratiometric measurements and FLIM. The metallic nanostructures will be used to measure metal ion distributions in cells with sub-wavelength resolution. Intensity ratio and lifetime images will be obtained by raster scanning the electric field arrays to obtain complete cellular metal ion images. Fluorescence microscopy is widely used in biology and cell imaging. Unfortunately, the spatial resolution is limited to about 300 nm, which is much larger than most biomolecules of interest. The goal of this project is to increase the spatial resolution to about 50 nm. The new microscope technology will be utilized by industry to develop new products.

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