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Bayesian localization microscopy reveals nanoscale podosome dynamics

Nature Methods volume 9, pages 195–200 (2012)Cite this article

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Abstract

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

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Figure 1: Correlative measurements using PALM imaging and Bayesian localization imaging on tubulin.
Figure 2: A 3B analysis of vinculin in fixed cells containing podosomes and labeled with Alexa 488.
Figure 3: Podosomes, visualized using an mCherry-tagged truncated talin construct, forming and dissociating in a live cell.
Figure 4: Dissociation and formation of groups of podosomes in a motile cell.
Figure 5: A 3B analysis of fixed-cell data to determine the colocalization of vinculin and the truncated talin construct in podosomes.
Figure 6: Simulations showing the performance of the 3B analysis method.

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Acknowledgements

We acknowledge helpful discussions with A. Fraser, F. Viola, P. Fox-Roberts, O. Mandula and J. Sleep. We thank M. Kielhorn for assistance in aligning the optical system and K. Glocer for critical reading of the manuscript. We thank M. Parsons (King's College London) for providing the template plasmid. We acknowledge support from the EU Seventh Framework Programme (FP7 Project GA 215597 (S.C. and R.H.), EU FP7 Project ITN 237946 T3Net (G.E.J.)), the Wellcome Trust (S.C., J.M. and G.E.J.), the Medical Research Council (UK) (G.E.J.) and the Royal Society (S.C.).

Author information

Author notes

  1. Susan Cox and Edward Rosten: These authors contributed equally to this work.

Authors and Affiliations

  1. Randall Division, King's College London, Guy's Campus, London, UK

    Susan Cox, James Monypenny, Gareth E Jones & Rainer Heintzmann

  2. Department of Engineering, University of Cambridge, Cambridge, UK

    Edward Rosten

  3. Computer Vision Consulting Ltd., Lynton House, Woking, Surrey, UK

    Edward Rosten

  4. National Institutes of Health, Cell Biology and Metabolism Branch, Bethesda, Maryland, USA

    Tijana Jovanovic-Talisman, Dylan T Burnette & Jennifer Lippincott-Schwartz

  5. Institute of Physical Chemistry, Friedrich-Schiller University Jena, Jena, Germany

    Rainer Heintzmann

  6. Institute of Photonic Technology, Jena, Germany

    Rainer Heintzmann

Authors
  1. Susan Cox
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  2. Edward Rosten
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  3. James Monypenny
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  4. Tijana Jovanovic-Talisman
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  5. Dylan T Burnette
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  7. Gareth E Jones
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Contributions

S.C., J.M., T.J.–T., D.T.B., J.L.-S., G.E.J. and R.H. conceived of and designed the experiments. S.C. and E.R. conceived of and designed the analysis. J.M. prepared the podosome samples, and T.J.–T. and D.T.B. prepared the samples for correlative measurements. S.C. and J.M. performed live-cell experiments, S.C. carried out fixed-cell experiments on podosomes, and T.J.–T. and D.T.B. carried out the correlative measurements. E.R. and S.C. carried out the data analysis and wrote the manuscript, and all authors revised the manuscript.

Corresponding author

Correspondence to Susan Cox.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Note (PDF 425 kb)

Supplementary Video 1

Raw data video of vinculin in fixed podosome samples. Samples were labeled with Alexa 488 and mounted in PBS with 100 mM mercaptoethanol added as a reducing agent to induce blinking. Sample was illuminated using a laser at 488nm with a nominal power of 1 kW/cm2. A series of 300 images were collected, taken at 50 frames per second. Scalebar is 500 nm. (MOV 766 kb)

Supplementary Video 2

Raw data video of live THP-1 cells stably expressing an mCherry tagged, truncated talin construct. Sample was illuminated with a Xenon arc lamp in the wavelength range 615-687 nm. Video shows the first 500 of 8,000 images taken at frame rates of 50 fps. Scalebar is 2 μm. (MOV 2552 kb)

Supplementary Video 3

Widefield (left) and 3B (right) video of a podosome being dissociated by unwinding. A truncated talin construct is labeled. This video corresponds to Figure 3a in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37C during imaging. Scalebar is 1 μm. (MOV 406 kb)

Supplementary Video 4

Widefield (left) and 3B (right) video of a podosome being dissociate by being drawn into its center. A truncated talin construct is labeled. This video corresponds to Figure 3b in the main text. Each widefield and 3B image is generated from 200 frames (4 s) of raw data, and are spaced 50 frames (1 s) apart. Widefield images are created by averaging. Cells were maintained at 37C during imaging. Scalebar is 1 μm. (MOV 577 kb)

Supplementary Video 5

Widefield (left) and 3B (right) video of podosomes being constructed. A truncated talin construct is labeled. This video corresponds to Figure 3c in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37C during imaging. Scalebar is 1 μm. (MOV 975 kb)

Supplementary Video 6

Widefield (left) and 3B (right) video of a steady state podosome in which a truncated talin construct is labeled. This video corresponds to Figure 3d in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37C during imaging. Scalebar is 500 nm. (MOV 343 kb)

Supplementary Video 7

Widefield (left) and 3B (right) video reveals podosomes in a motile cell to be highly dynamic. A truncated talin construct is labeled in these cells. This video includes, as a small part, the areas shown in Figure 4a–c in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 100 frames (2 s) apart. Widefield images are created by averaging. Cells were maintained at 37C during imaging. Scalebar is 2 μm. (MOV 5126 kb)

Supplementary Software

3B analysis software. Contains source code, test data and instructions for use. (ZIP 1681 kb)

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Cox, S., Rosten, E., Monypenny, J. et al. Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat Methods 9, 195–200 (2012). https://doi.org/10.1038/nmeth.1812

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