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.
Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA. burghardt@mayo.edu
Thomas P
TP
Burghardt
Jon E
JE
Charlesworth
Miriam F
MF
Halstead
James E
JE
Tarara
Katalin
K
Ajtai
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