Protocol | Published:

Superresolution live imaging of plant cells using structured illumination microscopy

Nature Protocols volume 10, pages 12481263 (2015) | Download Citation

Abstract

Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.

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Acknowledgements

Development of this protocol was financially supported by grant no. P501/11/1764 from the Czech Science Foundation (GACˇR); by grant no. LO1204 from the Czech Ministry of Education, Youth and Sports, National Program of Sustainability I (NPUI) to the Centre of the Region Haná for Biotechnological and Agricultural Research; and by the Czech National program of sustainability, LO1304.

Author information

Affiliations

  1. Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.

    • George Komis
    • , Olga Šamajová
    • , Miroslav Ovečka
    •  & Jozef Šamaj
  2. Institute of Molecular and Translational Medicine, Palacký University, Olomouc, Czech Republic.

    • Martin Mistrik
    •  & Jiri Bartek
  3. Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark.

    • Jiri Bartek

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Contributions

G.K., M.M., O.Š. and J.Š. performed the experiments. G.K., O.Š., M.M. and J.Š. established material preparation and microscope settings for SIM. M.M., O.Š., J.B. and J.Š. provided material and equipment. G.K. carried out quantitative analyses and validation procedures inherent to the protocol. M.O. prepared Supplementary Figure 1 with help from O.Š. and G.K. G.K. and J.Š. wrote the manuscript with editorial help from M.O., O.Š., M.M. and J.B. J.Š. and G.K. designed the experiments, and J.Š. supervised the whole project.

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2

Videos

  1. 1.

    Time-lapse imaging of cortical microtubule dynamics in a hypocotyl epidermal cell of Arabidopsis thaliana stably transformed with the GFP-MBD microtubule marker.

    Video corresponds to Fig. 7a,b. Recording time: 239.4 sec, Frames: 90, Time interval: 2.7 sec, Video frame rate: 16 fps

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DOI

https://doi.org/10.1038/nprot.2015.083

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