Surface Plasmon Assisted Microscope




Borejdo, Julian


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Total Internal Reflection Fluorescence (TIRF) microscopy is able to image 100-200 nm thick layer of a cell adjacent to a coverslip. However, it suffers from the fact that and excitation and emission light paths are shared. Here we suggest an alternative method of visualizing layer adjacent to a coverslip: a sample is put on a coverslip layered with a thin layer of gold and illuminated from the top, through water-based solution (Kretschmann illumination). The fluorophores near metal surface induce surface plasmons in the metal film. Fluorescence from these fluorophores couple with surface plasmons, permitting them to penetrate the metal. They emerge at Surface Plasmon Coupled Emission (SPCE) angle. The thickness of the observational layer is further reduced by metal quenching proximal (below 10 nm) to a surface. Fluorophores that are not proximal to a surface are unable to couple with the plasmons and are reflected back into the free space. High NA objective is used to collect image by EMCCD. Alternatively, the fluorescent signal is collcted by avalanche photodiode inserted in the conjugate image plane of the objective. The signal can be used to produce autocorrelation function of the motion of a sample and provide detailed information about its size and shape. Since the excitation and emission light paths are not shared, the system avoids problems associated through-the-objective TIRF detection. In addition, it the thickness of the observational volume is smaller than TIRF and has outstanding background rejection.