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