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Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amendable to automatically track non-plant structures with an apical growth.
Cytokinesis is the last step of cell division, when the mother cell cytoplasm is partitioned between two daughter cells (Lipka et al., 2015). In plants, it is achieved through the centrifugal expansion of a cell plate in the division plane, which eventually becomes the newly synthetized cell wall between the cells that underwent mitosis (Buschmann and Zachgo, 2016; Müller and Jürgens, 2016). Plant cells, being embedded in a stiff cell wall, cannot migrate. Orientation of cell division together with elongation is therefore critical for organ morphogenesis. Root meristems are a good model to study cell division because they are easily amenable to microscopy techniques without the need of dissection. However, roots undergoing cell division grow in length, and therefore require manual adjustment of the observation field over time. This protocol allows easy time-lapse imaging of cytokinesis, and of other cellular processes.
After making a movie of your stacks, you obtain stable image over time (Video 3).
We thank Dr. Laia Armengot and Dr. Antoine Larrieu for critical comments on the manuscript. M.D. is funded by a fellowship from the French Ministry of Higher Education and Research, Y.J. by ERC No. 3363360-APPL under FP/2007-2013, M-C.C by a group leader starting package «fond de recherche» from ENS Lyon.