In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy.
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 approximately 120 muM, implying single myosin detection volume for this biological assembly is approximately 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 approximately 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.
Burghardt
Thomas P
TP
Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA. burghardt@mayo.edu
Charlesworth
Jon E
JE
Halstead
Miriam F
MF
Tarara
James E
JE
Ajtai
Katalin
K
D000535:Aluminum / Q000737:chemistry*; D000818:Animals; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D007089:Image Enhancement / Q000379:methods*; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence / Q000379:methods*; D018485:Muscle Fibers, Skeletal / Q000648:ultrastructure*; D009218:Myosins / Q000648:ultrastructure*; D011817:Rabbits; D012518:Sarcomeres / Q000648:ultrastructure*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D000535:Aluminum; D009218:Myosins