SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
In Situ Fluorescent Protein Imaging with Metal Film Enhanced Total Internal Reflection Microscopy
Thomas P. Burghardt 1*, Jon E Charlesworth 2, Miriam F Halsetad 2, James E Tarara 2 and Katalin Ajtai 2
1 Mayo Foundation
2 Mayo
* To whom correspondence should be addressed. E-mail: burghardt{at}mayo.edu.
Submitted on December 8, 2005
Revised on December 21, 2005
Accepted on 3 March 2006
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Abstract |
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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 (Burghardt et al., 2006, Biochemistry, in press). Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface (Hellen & Axelrod, 1987, J. Opt. Soc. Am. B 4, 337-350). 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.
Key Words:
Fluorescence emission near an interface, Muscle fiber, Myosin cross-bridge, Near-field microscopy, Single molecule detection, Supercritical angle fluorescence
