DESCRIPTION (provided by applicant): This application addresses broad the Challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-GM-106; subcellular imaging of metal ions. Fluorescence microscopy is one of the most widely used methods in the biological sciences. However, the spatial resolution is limited to about 300 nm by the laws of diffractive optics. Methods to improve the 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. In this Challenge Grant application 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. Our approach is based on the emerging fields of plasmonic and nano-optics, on the novel optical properties of metallic nanostructures. It is known that sub- wavelength size electric field distributions can occur upon illumination of certain metallic nanostructures. Previous approaches to use these structures for imaging were based on contact of the sample with near-fields on the metal structure, typically within 50 nm. Such methods only allow measurements on the bottom contact region of the cell, which would need to be in contact with the metallic structure, and would only provide point measurements and not imaging. Contact-type microscopy cannot be used to image the intracellular ion concentration in cells. 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 photo bleaching. This Challenge Grant project is made possible by the availability of modern nanofabrication methods and computational methods. 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. We will refer to such structures as plasmonic lenses. The results from the FDTD simulations will be used to select specific geometries for nanofabrication. 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 beam (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). These fluorescence measurements will be performed using probes which are sensitive to metal ions including Na+, K+, Ca2+, Mg2+ and Zn2+. The known metal ion concentrations in the solution will be compared with the concentrations determined from the wavelength-radiometric measurements and FLIM. The metallic nanostructures will be used to measure metal ion distributions in cells with sub-wavelength resolution. We selected the different cell lines for imaging, including COS cells, 293 cells, PC12 cells and mitochondria therein. These cells have different properties and thus represent a range of imaging applications. Intensity ratio and lifetime images will be obtained by raster scanning the electric field arrays to obtain complete cellular metal ion images. The proposed plasmonic microscope will have a profound impact on research in the biosciences. These microscopes will be based on an inexpensive metallic structures and a CCD camera to record the images of the isolated spots. Simple raster scanning will be used to obtain the complete image. With or without sub-wavelength spatial resolution, such microscopes based on this novel imaging technique will find use not only in cell imaging, but also with high-throughput assays and medical diagnostics. This project is to develop a new type of microscope based on plasmonics and metallic nanostructures. 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, and thereby greatly increase the information available for cell imaging research. Development of the proposed microscope is a challenging goal. This project will stimulate the emission by the hiring of new scientists. The new microscope technology will be utilized by industry to develop new products.

This project is to develop a new type of microscope based on plasmonics and metallic nanostructures. 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, and thereby greatly increase the information available for cell imaging research. Development of the proposed microscope is a challenging goal. This project will stimulate the emission by the hiring of new scientists. The new microscope technology will be utilized by industry to develop new products.