High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particu...

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Autores principales: Braïni, Céline, Mottolese, Angelo, Ferrante, Ivan, Monnier, Sylvain, Villard, Catherine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933246/
https://www.ncbi.nlm.nih.gov/pubmed/29630044
http://dx.doi.org/10.3791/56923
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author Braïni, Céline
Mottolese, Angelo
Ferrante, Ivan
Monnier, Sylvain
Villard, Catherine
author_facet Braïni, Céline
Mottolese, Angelo
Ferrante, Ivan
Monnier, Sylvain
Villard, Catherine
author_sort Braïni, Céline
collection PubMed
description Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).
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spelling pubmed-59332462018-05-16 High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices Braïni, Céline Mottolese, Angelo Ferrante, Ivan Monnier, Sylvain Villard, Catherine J Vis Exp Neuroscience Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs). MyJove Corporation 2018-03-26 /pmc/articles/PMC5933246/ /pubmed/29630044 http://dx.doi.org/10.3791/56923 Text en Copyright © 2018, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Neuroscience
Braïni, Céline
Mottolese, Angelo
Ferrante, Ivan
Monnier, Sylvain
Villard, Catherine
High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title_full High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title_fullStr High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title_full_unstemmed High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title_short High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices
title_sort high-resolution volume imaging of neurons by the use of fluorescence exclusion method and dedicated microfluidic devices
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933246/
https://www.ncbi.nlm.nih.gov/pubmed/29630044
http://dx.doi.org/10.3791/56923
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