Reverse Kretchman Microscope

Reverse Kretchmann Microscope J. Borejdo1,2, R. Fudala1,2, 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 Purpose. The purpose of this research was to construct the instrument to supplement Total Internal Reflection Microscopy (TIRF) to mage 10-20 nm thick layer of cells such as membrane lipids, membrane receptors and other structures proximal to basal membranes. Methods. 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. Results. With little effort, we were able to image 100 nm fluorescent nanospheres. Conclusions. Reverse Kretchmann Microscope 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 surface