Bacterial adhesion to surfaces can lead to the formation of biofilms and the development of infection. Bacteria which are motile reach surfaces faster than nonmotile bacteria and may adhere more rapidly than nonmotile bacteria. The motility of a species has also been implicated as a factor in virulence. Understanding the role that motility plays in bringing a bacterium in contact with a surface and its subsequent adherence can aid in designing strategies to prevent adhesion. In this paper, we describe the development of a total internal reflection aqueous fluorescence (TIRAF) microscope to measure the distance between an E, coli bacterium and a clean quartz surface as the bacterium was swimming laterally along the surface. This technique is distinct from other related approaches such as atomic force microscopy and total internal reflection fluorescence microscopy because it does not require the immobilization of cells on a surface for the measurement. The TIRAF microscope was capable of capturing images of a field of bacteria two times per second for over 1 min. The analysis technique developed to translate the images into quantitative distance measurements, using the equations of Gingell, is also described. Both motile and nonmotile bacteria were observed within 100 nm of a clean quartz surface. TIRAF provided a quantitative measure of the distance between bacteria and a surface at nanometer scale resolution.