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 Light Sheet Fluorescence Microscopy of Calcium Oscillations in Arabidopsis thaliana / N. Romano Armada, F.G. Doccula, A. Candeo, G. Valentini, A. Costa, A. Bassi (METHODS IN MOLECULAR BIOLOGY). - In: Calcium Signalling : Methods and Protocols / [a cura di] A. Raffaello, D. Vecellio Reane. - [s.l] : Springer, 2019. - ISBN 9781493990177. - pp. 87-101

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

F.G. Doccula;A. Costa
Penultimo
Writing – Review & Editing
;
2019

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.
In vivo plant imaging; Light sheet fluorescent microscopy; Calcium oscillations; Cameleon; Calcium sensor; NES-YC3.6
Settore BIO/04 - Fisiologia Vegetale
2019
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/615694
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