Cargando…

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques...

Descripción completa

Detalles Bibliográficos
Autores principales: Mishra, Bibhudatta, Ghannad-Rezaie, Mostafa, Li, Jiaxing, Wang, Xin, Hao, Yan, Ye, Bing, Chronis, Nikos, Collins, Catherine A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117361/
https://www.ncbi.nlm.nih.gov/pubmed/24562098
http://dx.doi.org/10.3791/50998
_version_ 1782328688245735424
author Mishra, Bibhudatta
Ghannad-Rezaie, Mostafa
Li, Jiaxing
Wang, Xin
Hao, Yan
Ye, Bing
Chronis, Nikos
Collins, Catherine A.
author_facet Mishra, Bibhudatta
Ghannad-Rezaie, Mostafa
Li, Jiaxing
Wang, Xin
Hao, Yan
Ye, Bing
Chronis, Nikos
Collins, Catherine A.
author_sort Mishra, Bibhudatta
collection PubMed
description Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.
format Online
Article
Text
id pubmed-4117361
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher MyJove Corporation
record_format MEDLINE/PubMed
spelling pubmed-41173612015-02-07 Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae Mishra, Bibhudatta Ghannad-Rezaie, Mostafa Li, Jiaxing Wang, Xin Hao, Yan Ye, Bing Chronis, Nikos Collins, Catherine A. J Vis Exp Bioengineering Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush. MyJove Corporation 2014-02-07 /pmc/articles/PMC4117361/ /pubmed/24562098 http://dx.doi.org/10.3791/50998 Text en Copyright © 2014, 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 Bioengineering
Mishra, Bibhudatta
Ghannad-Rezaie, Mostafa
Li, Jiaxing
Wang, Xin
Hao, Yan
Ye, Bing
Chronis, Nikos
Collins, Catherine A.
Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title_full Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title_fullStr Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title_full_unstemmed Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title_short Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
title_sort using microfluidics chips for live imaging and study of injury responses in drosophila larvae
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117361/
https://www.ncbi.nlm.nih.gov/pubmed/24562098
http://dx.doi.org/10.3791/50998
work_keys_str_mv AT mishrabibhudatta usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT ghannadrezaiemostafa usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT lijiaxing usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT wangxin usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT haoyan usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT yebing usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT chronisnikos usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae
AT collinscatherinea usingmicrofluidicschipsforliveimagingandstudyofinjuryresponsesindrosophilalarvae