Nanometer distances between swimming bacteria and surfaces measured by total internal reflection aqueous fluorescence microscopy

Bacterial adhesion to surfaces can lead to the formation of biofilms and th e development of infection. Bacteria which are motile reach surfaces faster than nonmotile bacteria and may adhere more rapidly than nonmotile bacteri a. The motility of a species has also been implicated as a factor in virule nce. Understanding the role that motility plays in bringing a bacterium in contact with a surface and its subsequent adherence can aid in designing st rategies to prevent adhesion. In this paper, we describe the development of a total internal reflection aqueous fluorescence (TIRAF) microscope to mea sure the distance between an E, coli bacterium and a clean quartz surface a s 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 requ ire the immobilization of cells on a surface for the measurement. The TIRAF microscope was capable of capturing images of a field of bacteria two time s 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 observe d within 100 nm of a clean quartz surface. TIRAF provided a quantitative me asure of the distance between bacteria and a surface at nanometer scale res olution.