Advertisement

In Vivo Light Sheet Fluorescence Microscopy of Calcium Oscillations in Arabidopsis thaliana

  • Neli Romano Armada
  • Fabrizio Gandolfo Doccula
  • Alessia Candeo
  • Gianluca Valentini
  • Alex CostaEmail author
  • Andrea Bassi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)

Abstract

Calcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant’s growth and physiological conditions.

A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs.

Key words

In vivo plant imaging Light sheet fluorescent microscopy Calcium oscillations Cameleon Calcium sensor NES-YC3.6 

Notes

Acknowledgments

This work was supported by Laserlab-Europe [EU-H2020 654148] and by Università degli Studi di Milano [PIANO DI SVILUPPO DI ATENEO 2016] to A.Co. NRA was a beneficiary of a fellowship from the European Commission within the framework of the “SUSTAIN-T Project of the Erasmus Mundus Programme, Action 2—STRAND 1, Lot 7, Latin America.”

References

  1. 1.
    Clapham DE (2007) Calcium signaling. Cell 131:1047–1058CrossRefGoogle Scholar
  2. 2.
    Allen GJ, Kwak JM, Chu SP et al (1999) Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J 19:735–747CrossRefGoogle Scholar
  3. 3.
    Krebs M, Held K, Binder A et al (2012) FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+ dynamics. Plant J 69:181–192CrossRefGoogle Scholar
  4. 4.
    Loro G, Drago I, Pozzan T et al (2012) Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells. Plant J 71:1–13CrossRefGoogle Scholar
  5. 5.
    Maizel A, von WD, Federici F et al (2011) High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant J 68:377–385CrossRefGoogle Scholar
  6. 6.
    Sena G, Frentz Z, Birnbaum KD et al (2011) Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy. PLoS One 6:e21303CrossRefGoogle Scholar
  7. 7.
    Costa A, Candeo A, Fieramonti L et al (2013) Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy. PLoS One 8:e75646CrossRefGoogle Scholar
  8. 8.
    Candeo A, Doccula FG, Valentini G et al (2017) Light sheet fluorescence microscopy quantifies calcium oscillations in root hairs of Arabidopsis thaliana. Plant Cell Physiol 58:1161–1172CrossRefGoogle Scholar
  9. 9.
    Zagato E, Toon B, De Smedt SC et al (2018) Technical implementations of light sheet microscopy. Microsc Res Tech:1–18Google Scholar
  10. 10.
    Bassi A, Schmid B, Huisken J (2015) Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development. Development 142:1016–1020CrossRefGoogle Scholar
  11. 11.
    Nagai T, Yamada S, Tominaga T et al (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559CrossRefGoogle Scholar
  12. 12.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  13. 13.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar
  14. 14.
    Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Neli Romano Armada
    • 1
    • 2
  • Fabrizio Gandolfo Doccula
    • 3
  • Alessia Candeo
    • 1
  • Gianluca Valentini
    • 1
    • 4
  • Alex Costa
    • 3
    Email author
  • Andrea Bassi
    • 1
    • 4
  1. 1.Dipartimento di FisicaPolitecnico di MilanoMilanItaly
  2. 2.INIQUI y Facultad de IngenieríaUniversidad Nacional de SaltaSaltaArgentina
  3. 3.Dipartimento di BioscienzeUniversità degli studi di MilanoMilanItaly
  4. 4.Istituto di Fotonica e NanotecnologieConsiglio Nazionale delle RicercheMilanItaly

Personalised recommendations