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In Situ Fluorescent Protein Imaging with Metal Film-Enhanced Total Internal Reflection Microscopy

Thomas P. Burghardt ^*, Jon E. Charlesworth , Miriam F. Halstead ^*, James E. Tarara  and Katalin Ajtai ^*

^* Department of Physiology and Biomedical Engineering, Electron Microscope Laboratory, and Confocal Microscopy Laboratory, Mayo Clinic Rochester, Rochester, Minnesota 55905

Correspondence: Address reprint requests to Thomas P. Burghardt, E-mail: burghardt{at}mayo.edu.

Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of 120 µM, implying single myosin detection volume for this biological assembly is 1 attoL (10^–18 L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from 3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM's usefulness for single molecule isolation within biological assemblies.

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