TIRF microscopy evanescent field calibration using tilted fluorescent microtubules

Research output: Contribution to journalResearch articleContributedpeer-review


  • C. Gell - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • M. Berndt - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • J. Enderlein - , University of Göttingen (Author)
  • S. Diez - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)


Summary 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.


Original languageEnglish
Pages (from-to)38-46
Number of pages9
JournalJournal of microscopy : principal publication of the Royal Microscopical Society
Issue number1
Publication statusPublished - Apr 2009
Externally publishedYes

External IDs

PubMed 19335455
ORCID /0000-0002-0750-8515/work/142235573



  • Evanescent field, Microscope calibration, Microtubules, Penetration depth, TIRF

Library keywords