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Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells

Nature Cell Biology volume 3, pages 852–855 (2001)Cite this article

Abstract

Live-cell imaging technology using fluorescent proteins (green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics1,2. But tools that can quantitatively analyse complex spatiotemporal processes in live cells remain lacking. Here we describe a new technique — fast multi-colour four-dimensional imaging combined with automated and quantitative time-space reconstruction — to fill this gap. As a proof of principle, we apply this method to study the re-formation of the nuclear envelope in live cells3,4,5,6. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution. The highly complex data, comprising several thousand images from a single cell, were quantitatively reconstructed in time–space by software developed in-house. This analysis reveals quantitative and qualitative insights into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion — results that would have been impossible to achieve by manual inspection alone. Our new technique will greatly facilitate study of the highly ordered dynamic architecture of eukaryotic cells.

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Figure 1: Three-dimensional reconstruction of LBR–YFP recruitment to patches on chromatin surface.
Figure 2: High-speed 4D imaging of early LBR patch formation on chromatin surface.
Figure 3: Synchronized expansion of nuclear envelope and chromatin domain.

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Acknowledgements

We thank B. Oakley for the γ-tubulin cDNA; N. Daigle for technical assistance; J. Mattes for providing the matching algorithm; and C. Conrad and C. Athale for critical comments on the manuscript. The bioinformatics group acknowledges the support of the German Federal Ministry of Education and Research (BMBF) through the BioFuture grant (AZ 11880A) and of the German Research Council (Ei 358/2-1 and Ei 358/1-1). J.B. was supported by a fellowship through EMBL's international Ph.D. programme. Part of this work was performed in collaboration with T.I.L.L.-Photonics, Munich, Germany.

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Authors and Affiliations

  1. Intelligent Bioinformatics Systems Department, German Cancer Research Centre, Heidelberg, 69120, Germany

    Daniel Gerlich, Matthias Gebhard & Roland Eils

  2. Gene Expression and Cell Biology/Biophysics Programmes, EMBL, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Joël Beaudouin & Jan Ellenberg

Authors
  1. Daniel Gerlich
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  3. Matthias Gebhard
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  4. Jan Ellenberg
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  5. Roland Eils
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Corresponding author

Correspondence to Roland Eils.

Supplementary information

Supplementary figures

Figure S1 Detailed description of 3-D reconstruction technique (PDF 317 kb)

Figure S2 Integration of LBR into patches and the NE measured in single slices versus 4-D analysis

Figure S3 Comparison of volume expansion quantified with 4-D methods versus 2-D analysis in projected image stacks

Figure S4 Reliability of 4-D quantification with threshold segmentation

Supplementary movie

Movie 1 800 1000 Movie 1 Assembly of image stack from optical slices and reconstruction of a 3-D surface model of LBR-EYFP patches (green) on chromatin surface (red). First, the pre-processed optical image slices of a single image stack are displayed sequentially. Then, they are removed stepwise to reveal the 3-D reconstructed surface model. For further details see fig. 1. (MOV 285 kb)

Supplementary movie

Movie 2 Animated sequence of 3-D reconstructions of early LBR patch formation on chromatin surface. For details see fig. 2. (MOV 1659 kb)

Supplementary movie

Movie 3 Morphed reconstruction of NE and chromatin expansion. Four intermediate states of chromatin and NE surfaces per time frame were interpolated, thereby increasing temporal resolution. For details see fig. 3. (MOV 1022 kb)

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Gerlich, D., Beaudouin, J., Gebhard, M. et al. Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells. Nat Cell Biol 3, 852–855 (2001). https://doi.org/10.1038/ncb0901-852

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