Evanescent excitation microscopy. Its application to the study of single molecular process kinetics of actomyosin...

Evanescent field was generated on the stage of an inverted microscope upon an incidence of 532 nm Nd-YAG laser beam on interface between aqueous solution and fused silica glass. Thick filaments isolated from Mytilus edulis were adsorbed to the glass surface and nanomolar concentration of adenosine triphosphate (ATP) labeled with rhodamine was allowed to interact with thick filaments. The fluorescence from the surface was observed by triple-view microscopy at video rate. There were many fluorescent spots at the interface, which we identified as individual fluorescent ATP molecules. We found that the fluorescence from those spots was polarized. Fluorescence intensity of individual spots fluctuated considerably. We interpret the latter observation as a result of change in the orientation of emission dipole of the fluorescent ATP analog.