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Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of f...

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Autores principales: Dubey, Shiv Mani, Fendrych, Matyáš, Serre, Nelson Bc
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Bio-Protocol 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10367083/
https://www.ncbi.nlm.nih.gov/pubmed/37497461
http://dx.doi.org/10.21769/BioProtoc.4778
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author Dubey, Shiv Mani
Fendrych, Matyáš
Serre, Nelson Bc
author_facet Dubey, Shiv Mani
Fendrych, Matyáš
Serre, Nelson Bc
author_sort Dubey, Shiv Mani
collection PubMed
description In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC(2)(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC(2)(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.
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spelling pubmed-103670832023-07-26 Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots Dubey, Shiv Mani Fendrych, Matyáš Serre, Nelson Bc Bio Protoc Methods Article In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC(2)(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC(2)(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification. Bio-Protocol 2023-07-20 /pmc/articles/PMC10367083/ /pubmed/37497461 http://dx.doi.org/10.21769/BioProtoc.4778 Text en ©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
spellingShingle Methods Article
Dubey, Shiv Mani
Fendrych, Matyáš
Serre, Nelson Bc
Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title_full Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title_fullStr Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title_full_unstemmed Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title_short Relative Membrane Potential Measurements Using DISBAC(2)(3) Fluorescence in Arabidopsis thaliana Primary Roots
title_sort relative membrane potential measurements using disbac(2)(3) fluorescence in arabidopsis thaliana primary roots
topic Methods Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10367083/
https://www.ncbi.nlm.nih.gov/pubmed/37497461
http://dx.doi.org/10.21769/BioProtoc.4778
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