Abstract
Observing cellular and molecular processes in living organisms is key for understanding many important biological processes. Confocal microscopy is excellently suited for this as it enables the observation of molecules and cells in tissue layers of living organisms in three dimensions over time. However, in continuously growing organs, such as plant roots, observations over extended time spans become difficult as the specimen quickly grows out the field of view. Here, we provide a protocol that allows for the acquisition of confocal microscope time-lapse images of root tips spanning many hours, as the growing root tip is tracked and the microscopy is automatized to change the position of the stage. Importantly, due to its specific setup, this protocol allows for observing the effects of chemical stimuli or for creating specific growth conditions by precisely defining the growth medium during imaging. The protocol is suitable for observing multiple fluorophores, thereby moving beyond the level of individual genes. It is also simple enough to conduct larger numbers of these assays. Here we exemplify our method by describing the observation of root growth and GFP intensity in root tips under iron depletion conditions.
References
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Acknowledgment
We wish to thank all Busch lab members for support and helpful discussions, especially Ceren Tabak for taking pictures of the confocal chamber preparation. We thank Matt Watson for manuscript editing. This work was supported by funds from the Austrian Academy of Sciences through the Gregor Mendel Institute and an FWF stand-alone project (P27163-B22).
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Stoeva, D., Göschl, C., Corliss, B., Busch, W. (2017). Long-Term Confocal Imaging of Arabidopsis thaliana Roots for Simultaneous Quantification of Root Growth and Fluorescent Signals. In: Busch, W. (eds) Plant Genomics. Methods in Molecular Biology, vol 1610. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7003-2_12
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DOI: https://doi.org/10.1007/978-1-4939-7003-2_12
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