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

Nature Protocols volume 10, pages 1234–1247 (2015)Cite this article

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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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Figure 1: Basic description of the light-sheet microscopy and stage for sample insertion and imaging.
Figure 2: Preparation of A. thaliana seeds for light-sheet microscopy imaging of seed germination and early plant development.
Figure 3: Preparation of A. thaliana seedlings for light-sheet microscopy imaging.
Figure 4: Open system for the preparation of A. thaliana seeds and seedlings for light-sheet microscopy.
Figure 5: Arabidopsis thaliana seedlings mounted in the open system for imaging of lateral root development.
Figure 6: Time-lapse imaging of growing seedlings using light-sheet microscopy.
Figure 7: Light-sheet microscopy imaging of microtubules in etiolated seedlings of A. thaliana carrying the fluorescent microtubule marker GFP-MBD.
Figure 8: Simultaneous imaging of microtubules and F-actin in cotyledon cells of A. thaliana by dual-channel light-sheet microscopy.

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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.

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Authors and 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

Authors
  1. Miroslav Ovečka
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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.

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Correspondence to Jozef Šamaj.

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Supplementary information

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. (MOV 2181 kb)

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). (MOV 7082 kb)

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. (MOV 9677 kb)

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). (MOV 10000 kb)

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. (MOV 4093 kb)

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. (MOV 11773 kb)

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. (MOV 542 kb)

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. (AVI 11256 kb)

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. (AVI 11609 kb)

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Ovečka, M., Vaškebová, L., Komis, G. et al. Preparation of plants for developmental and cellular imaging by light-sheet microscopy. Nat Protoc 10, 1234–1247 (2015). https://doi.org/10.1038/nprot.2015.081

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