Characterization of the Evanescent Field in Objective-Based Total-Internal-Reflection Fluorescence (TIRF) Microscopy

Owing to its single-molecule sensitivity, objective-based total-internal-reflection fluorescence microscopy has been used to study various biological phenomena, such as conformation changes of a single biomolecule and localization and movement of single molecules inside a cell. In the technique, fluorophores associated with biomolecules are excited by an evanescent field formed near the water/coverglass interface (typically, within 100 to 200 nm). Here, we characterized the axial profile of the evanescent field. By using a prism-based scheme and applying Snell's law, we experimentally measured the incident angle of the excitation light and calculated the penetration depth of the evanescent field. The penetration depth was also acquired experimentally by measuring the fluorescence from a fluorescent bead for the excitation light at various incident angles. These independent approaches permitted highly accurate characterization of the evanescent field profile. This field profile provided the information on the distribution of the evanescent field along the axial direction, which will be useful in studying with this technique various biological phenomena as it provides a means to probe the axial dimension precisely.