title: TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.
abstract: Total internal reflection fluorescence microscopy has become a powerful tool to study the dynamics of sub-cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.
authors.affiliation: Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Gell
authors.forename: M
authors.initials: M
authors.lastname: Berndt
authors.forename: J
authors.initials: J
authors.lastname: Enderlein
authors.forename: S
authors.initials: S
authors.lastname: Diez