Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy.

Imaging of single fluorescent molecules has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single molecules of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 x, NA 1.4) with ordinary coverslips and immersion oil. 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 biologically active molecules. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single molecules.