These authors contributed equally to this work.
TIRF microscopy evanescent field calibration using tilted fluorescent microtubules
Article first published online: 19 MAR 2009
© 2009 The Authors Journal compilation © 2009 The Royal Microscopical Society
Journal of Microscopy
Volume 234, Issue 1, pages 38–46, April 2009
How to Cite
GELL, C., BERNDT, M., ENDERLEIN, J. and DIEZ, S. (2009), TIRF microscopy evanescent field calibration using tilted fluorescent microtubules. Journal of Microscopy, 234: 38–46. doi: 10.1111/j.1365-2818.2009.03147.x
- Issue published online: 19 MAR 2009
- Article first published online: 19 MAR 2009
- Received 4 October 2008; accepted 7 November 2008
- Evanescent field;
- microscope calibration;
- penetration depth;
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.