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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