Abstract

Single molecule observations usually require concentrations in the picomolar to nanomolar range. Such low concentrations guarantee that, on average, only a few molecules or less will be present within the focal volume of a high numerical aperture objective. The functional dynamics of a single enzyme molecule can be observed by immobilizing the enzyme in the presence of freely diffusing, fluorescent substrates. In this case, reducing the number of observed substrate molecules suppresses background fluorescence, allowing detection of the activity of the single enzyme through its effect on individually observed substrate molecules. For many biologically relevant systems, however, micromolar concentrations of ligand are necessary for proper enzyme function.

© 2002 Optical Society of America