Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning mic
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(2019) 15:120 Tofanelli et al. Plant Methods https://doi.org/10.1186/s13007-019-0505-x
Open Access
METHODOLOGY
Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy Rachele Tofanelli1†, Athul Vijayan1†, Sebastian Scholz1,2 and Kay Schneitz1*
Abstract Background: A salient topic in developmental biology relates to the molecular and genetic mechanisms that underlie tissue morphogenesis. Modern quantitative approaches to this central question frequently involve digital cellular models of the organ or tissue under study. The ovules of the model species Arabidopsis thaliana have long been established as a model system for the study of organogenesis in plants. While ovule development in Arabidopsis can be followed by a variety of different imaging techniques, no experimental strategy presently exists that enables an easy and straightforward investigation of the morphology of internal tissues of the ovule with cellular resolution. Results: We developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide. We further improved the microscopy by employing a homogenous immersion system aimed at minimizing refractive index differences. The method allows complete inspection of the cellular architecture even deep within the ovule. Using the new protocol we were able to generate digital three-dimensional models of ovules of various stages. Conclusions: The protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages. From the imaging data three-dimensional digital ovule models with cellular resolution can be rapidly generated using image analysis software, for example MorphographX. Such digital models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species. Keywords: 3D reconstruction, 3D organ models, Arabidopsis, ClearSee, Imaging, Ovule, SCRI Renaissance 2200, To-PRO-3 iodide Background The ovule is the major female plant organ involved in sexual reproduction and the progenitor of the seed. In part due to their highly interesting biology the ovules of Arabidopsis thaliana also successfully serve as a general model system to study the molecular and genetic basis of plant organogenesis [1–5].
*Correspondence: [email protected] † Rachele Tofanelli and Athul Vijayan contributed equally to this work 1 Entwicklungsbiologie der Pflanzen, Wissenschaftszentrum Weihenstephan, Technische Universität München, Emil‑Ramann‑Str. 4, 85354 Freising, Germany Full list of author information is available at the end of the article
An individual Arabidopsis ovule primordium emerges as a finger-like protrusion emanating from the placental tissue of the gynoecium [6, 7]. Shortly thereafter, three distinct pattern elements can be recognized al
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