Protocol | Published:

Preparation of plants for developmental and cellular imaging by light-sheet microscopy

Nature Protocols volume 10, pages 12341247 (2015) | Download Citation

Abstract

Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.

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Acknowledgements

This work was supported by grant no. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, Ministry of Education, Youth and Sports, Czech Republic.

Author information

Affiliations

  1. Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic.

    • Miroslav Ovečka
    • , Lenka Vaškebová
    • , George Komis
    • , Ivan Luptovčiak
    •  & Jozef Šamaj
  2. Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA.

    • Andrei Smertenko

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Contributions

J.Š. and M.O. conceived the project and designed the experiments, and evaluated the data and wrote the paper with the input of all other authors. M.O., L.V., G.K., I.L. and A.S. performed experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jozef Šamaj.

Supplementary information

Videos

  1. 1.

    Seed plating for the open system

    Instructive movie of seed plating for sample preparation in the open system without piston. Depression of the surface of ½ MS medium solidified with 0.6% (wt/vol) Phytagel using sterile pipette tip and placing sterilized seeds of A. thaliana to these depressions.

  2. 2.

    Seedling insertion into FEP tube

    Instructive movie of seedling insertion into an open FEP tube with an inner diameter of 1.1 mm (without piston).

  3. 3.

    FEP tube containing seedling

    Overview of the seedling inserted in open FEP tube with inner diameter of 1.1 mm (without piston), fixed in green-labeled capillary.

  4. 4.

    Large seedling insertion into FEP tube

    Instructive movie showing large seedling insertion into an open FEP tube with an inner diameter of 2.8 mm (without piston).

  5. 5.

    FEP tube containing large seedling

    Overview of the seedling inserted in open FEP tube with an inner diameter of 2.8 mm (without piston), fixed in blue-labeled capillary.

  6. 6.

    Seed germination

    Time-lapse movie showing seed germination of A. thaliana transgenic line carrying fluorescent microtubule marker GFP-TUA6. Seed was embedded in ½ MS medium in the FEP tube, which was plugged by 1% (wt/vol) low gelling temperature agarose. Recording time of 5 h and 45 min, frame acquired every 5 min, 68 frames in total, video rate of 18 fps.

  7. 7.

    Seed germination and primary root growth

    Time-lapse movie showing seed germination and primary root growth of A. thaliana transgenic line carrying fluorescent microtubule marker GFP-TUA5. Plant growing in ½ MS medium solidified with Phytagel was prepared for imaging in the open system (without piston) using an FEP tube with inner diameter of 1.1 mm fixed in the green-labeled capillary. Recording time of 26 h and 40 min, frames acquired every 10 min, 158 frames in total, video rate of 18 fps.

  8. 8.

    Lateral root growth

    Time-lapse movie showing lateral root formation in A. thaliana line carrying fluorescent microtubule marker GFP-TUA5. Plant growing in ½ MS medium solidified with Phytagel was prepared for imaging in the open system (without piston). After seedling enclosing by FEP tube with inner diameter of 2.8 mm in Petri plate for 3 days, FEP tube with sample was removed and fixed to the blue- labeled capillary. Recording time of 48 h, frames acquired every 20 min, 144 frames in total, video rate of 10 fps.

  9. 9.

    Actin cytoskeleton

    Time-lapse movie of actin cytoskeleton in cotyledon epidermal cells of light-grown A. thaliana seedling carrying fluorescent F-actin marker FABD2-GFP. Sample was embedded in 1% (wt/vol) low gelling temperature agarose in the glass capillary. Recording time of 4 min and 48 s, frame acquired every 5.8 s, 50 frames in total, video rate of 18 fps.

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DOI

https://doi.org/10.1038/nprot.2015.081

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