(1) Single Molecule Imaging of Enzymatic Reactions
Using Objective-type Evanescent Illumination

Makio TOKUNAGA, Kiwamu SAITO¹, Kazuo KITAMURA², Atsuko IWANE² and Toshio YANAGIDA² (¹Department of Physics, Kanazawa University, ²Department of Physiology I, Osaka University Medical School)

Imaging of single fluorescent molecules has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). This method allowed visualization of single molecules under scanning probe microscopes. Taking advantage of the technique of single molecule imaging, individual ATP turnovers have been visualized with a fluorescent ATP analogue, Cy3-ATP, using a simple experimental strategy. Clear on/off signals were obtained that correspond to the association and dissociation of single Cy3-ATP/ADP molecules with a single myosin head molecule. This method will allow a variety of single-molecular assays of biomolecular functions to be performed using fluorescently labeled substrates, ligands, messengers and so on. For details, see Ref. 4.