These authors contributed equally to this work.
TIRF microscopy evanescent field calibration using tilted fluorescent microtubules
Version of Record 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 online: 19 MAR 2009
- Version of Record online: 19 MAR 2009
- Received 4 October 2008; accepted 7 November 2008
Fig. S1. The normalized intensity profile along a titled microtubule measured in TIRF for a laser AOI of 63°, before (red) and after (blue) multiple measurements at other AOIs. These data show that the reproducibility of the AOI is good and that over the time course of a typical measurement neither bleaching nor microtubule depolymerization are significant effects.
Fig. S2. Intensity profiles along a steeply tilted (approximately 15.9°) microtubule in both uniform illumination (a laser beam at 0° AOI) and with EPI illumination are nearly identical.
Fig. S3. Method to determine microtubule tilt angle: A z stack (50 nm resolution) of a tilted microtubule was recorded using EPI illumination. Maximum projections were then made in the three orthogonal directions; shown above are the A): z projection, B): x projection and C): y projection. Recording the microtubule angle with respect to two orthogonal axes allows calculation of the ‘apparent’ tilt angle with respect to the cover glass. D) The microtubule's central axis is located by multiple Lorentzian fits along the microtubule (three such fits are shown for illustration, which are the line profiles and fits along the similarly coloured lines shown in C). E) The slope of a plot of the microtubule z position (Lorentzian peak tops) versus position along the microtubule (E shows the plot for the image in C) gives the tangent of the microtubule angle from that projection.
Appendix S1. Supplementary information
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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.