Surface Plasmon Assisted Microscope (SPAM)

Surface Plasmon Assisted Microscope (SPAM) J. Borejdo1,2, Z. Gryczynski3, R. Fudala1,2, C. Joshi1, K. Borgmann1, A. Ghorpade1 and I. Gryczynski1,2 1 Dept of Microbiology, Immunology and Genetics, University of North Texas, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107 2Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107 3 Dept of Physics and Astronomy, Texas Christian University, 2800 S. University Dr., Fort Worth, Texas 76129 The purpose of this research was to construct the instrument to image 10-20 nm thick layer of cells such as membrane lipids, membrane receptors and other structures proximal to basal membranes. Until now this task was best achieved by Total Internal Reflection Microscopy (TIRF). Here we demonstrate an alternative method which has number of advantages over TIRF. The materials required for constructing such an instrument include inverted microscope, a high refractive index coverslip covered with 50 nm thick layer of gold and an optical fiber coupled laser. A sample is placed on a high refractive index coverslip coated with a metal instead of glass and is illuminated by the laser from the top (through aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a Surface Plasmon Coupled Emission (SPCE) angle. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single mode optical fiber inserted in the conjugate image plane of the objective. We compared images of cell monolayer of astrocytes transfected with GFP obtained by SPAM with image obtained by TIRF. We think that SPAM image was clearer than TIRF image because: 1. Thickness of the detection layer was reduced in comparison with TIRF because metal quenched fluorophores at a close proximity (below 10 nm) to a surface; 2. The system avoided complications of through-the-objective TIRF associated with shared excitation and emission light path; 3. The microscope had excellent background rejection because all far-field radiation is reflected by the mirror-like metal layer.