title: Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana.
abstract: Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae and algal rhizoids). High-resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that light sheet fluorescence microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosol-localized ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterized their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs.
authors.affiliation: Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Candeo
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Giovanni Celoria 26, 20133 Milano, Italy.
authors.forename: Fabrizio G
authors.initials: FG
authors.lastname: Doccula
authors.affiliation: Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Valentini
authors.affiliation: Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bassi
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Giovanni Celoria 26, 20133 Milano, Italy.
authors.forename: Alex
authors.initials: A
authors.lastname: Costa
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000254:growth & development / Q000502:physiology; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D018517:Plant Roots / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D002147:Calmodulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C579281:YCaM3.6 protein
keywords: Arabidopsis thaliana; Calcium oscillations; Cameleon calcium probe; Light sheet fluorescence microscopy; Root hairs