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Three-dimensional preparation and imaging reveal intrinsic microtubule properties

Nature Methods volume 4, pages 843–846 (2007)Cite this article

Abstract

We present an experimental investigation of microtubule dynamic instability in three dimensions, based on laser light-sheet fluorescence microscopy. We introduce three-dimensional (3D) preparation of Xenopus laevis egg extracts in Teflon-based cylinders and provide algorithms for 3D image processing. Our approach gives experimental access to the intrinsic dynamic properties of microtubules and to microtubule population statistics in single asters. We obtain evidence for a stochastic nature of microtubule pausing.

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Figure 1: Measurement of 3D microtubule dynamic instability.
Figure 2: Side-by-side comparison of 2D and 3D experiments of microtubule dynamic instability.
Figure 3: 2D and 3D microtubule growth speeds distributions and analysis of growth speed contrast.
Figure 4: T population statistics and its application in the analysis of microtubule pausing.

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Acknowledgements

We thank K. Greger for implementing the high-resolution illumination system, and J. Huisken and J. Swoger for helpful comments on SPIM and 3D image processing. We are grateful to I. Kronja, J. Cahu, A. Kruljac-Letunic, M. Caudron, S. Kandels-Lewis and E. Karsenti (Cell Biology and Biophysics Unit, EMBL) for providing reagents and advice on X. laevis egg extracts. We thank H. Kress, D. Brunner, T. Surrey, M. Knop and E. Karsenti for critical comments on the manuscript.

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Authors and Affiliations

  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Philipp J Keller, Francesco Pampaloni & Ernst H K Stelzer

Authors
  1. Philipp J Keller
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  2. Francesco Pampaloni
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  3. Ernst H K Stelzer
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Contributions

P.J.K. developed the sample preparation in Teflon-based cylinders, performed the experiments, developed the image processing and data analysis algorithms and wrote most of the paper. F.P. trained P.J.K. in the biochemical aspects of the experiments and participated in project planning, in the 2D experiments and in manuscript preparation. E.H.K.S. outlined and supervised the project and participated in all stages of manuscript preparation.

Corresponding authors

Correspondence to Philipp J Keller or Ernst H K Stelzer.

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Competing interests

The authors applied for a patent on the sample preparation in transparent Teflon-based cylinders.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1, Supplementary Methods (PDF 2339 kb)

Supplementary Video 1

Experimental arrangement at the SPIM imaging chamber. The movie shows an illustration of the SPIM imaging chamber, the sample cylinder containing the egg extract (indicated in yellow), the illuminating light sheet (indicated in blue) and the detection objective. The light sheet is coplanar with the focal plane of the detection objective. (MOV 2189 kb)

Supplementary Video 2

Schematic illustration of the 3D imaging of a MT aster with SPIM. The aster's 3D structure is indicated in yellow. The Teflon-based cylinder's wall is shown in light grey. The illuminating light sheet (blue) enters the sample cylinder and excites the fluorescently-labelled tubulin molecules (fluorescence highlighted in green). The fluorescence signal is particularly strong in regions with a high tubulin density, i.e. for the intersecting volumes of microtubules with the light sheet (light green). By moving the sample through the light sheet and continuously recording images with a CCD camera, a 3D data set of the microtubule aster is recorded. Inset: Image stack showing a microtubule aster in Xenopus laevis egg extract. The image stack represents an early time point of a SPIM time-lapse data set of microtubule dynamics. Recording of the set of images took four seconds. Tubulin was labelled with Alexa-488. Fluorescence was detected via a Zeiss Achroplan 100x/1.0 water-dipping objective. (MOV 1615 kb)

Supplementary Video 3

3D MT dynamics recorded with SPIM. The movie shows maximum intensity projections along the axis of detection for a time-lapse data set of microtubule dynamics. Each projection is based on 28 planes that were recorded with a spacing of 700 nm. The time spacing between subsequent recordings of the entire 3D volume is 3.55 sec. Tubulin was labelled with Alexa-488. Fluorescence was detected via a Zeiss Achroplan 100x/1.0 water-dipping objective. (MOV 3290 kb)

Supplementary Video 4

Interphasic MT aster digitalized with “processTL”. The animation shows a visualization of the 3D traces of an interphasic aster's MTs at one time point. The data set was recorded with SPIM and processed by the automatic image processing algorithm “processTL”. The blue dots highlight the surface of the mapping sphere that is employed to detect initial MT intersections. These intersections are indicated as crosses on the surface (either in red or green). Green and red dots correspond to the detected contour points on the MTs. The MTs' plus-ends and minus-ends are also highlighted by crosses. Green traces indicate MTs that were partly located outside the recording volume. For those traces the last cross does not correspond to the plus-end, but rather to the position at which the tracing algorithm recognized an intersection of the MT's contour with the recording volume's boundaries. Red traces indicate MTs that were located fully inside the recording volume. (MOV 2833 kb)

Supplementary Software

Software package for image processing of 2D and 3D time-lapse data of dynamic MT asters. (ZIP 137 kb)

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Keller, P., Pampaloni, F. & Stelzer, E. Three-dimensional preparation and imaging reveal intrinsic microtubule properties. Nat Methods 4, 843–846 (2007). https://doi.org/10.1038/nmeth1087

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