Dual-colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti-Stokes fluorescence.
We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine-5-iodoacetamide interacting with a BODIPY FL-labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3-labelled ATP by single myosin molecules and sliding of a BODIPY FL-labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.
Saito
K
K
Yanagida Biomotron Project, ERATO. JST, Osaka, Japan. saito@yanagida.jst.go.jp
Tokunaga
M
M
Iwane
A H
AH
Yanagida
T
T
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000205:Actomyosin / Q000378:metabolism / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001896:Boron Compounds / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018715:Microscopy, Video; D015879:Myosin Subfragments / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000199:Actins; D001896:Boron Compounds; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D012235:Rhodamines; C089187:cyanine dye 3; D000255:Adenosine Triphosphate; D000205:Actomyosin