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Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2009
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726581/ https://www.ncbi.nlm.nih.gov/pubmed/19229185 http://dx.doi.org/10.3791/1129 |
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author | O'Brien, Georgeann S. Rieger, Sandra Martin, Seanna M. Cavanaugh, Ann M. Portera-Cailliau, Carlos Sagasti, Alvaro |
author_facet | O'Brien, Georgeann S. Rieger, Sandra Martin, Seanna M. Cavanaugh, Ann M. Portera-Cailliau, Carlos Sagasti, Alvaro |
author_sort | O'Brien, Georgeann S. |
collection | PubMed |
description | Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples. Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter (1). We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. |
format | Text |
id | pubmed-2726581 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-27265812011-07-21 Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos O'Brien, Georgeann S. Rieger, Sandra Martin, Seanna M. Cavanaugh, Ann M. Portera-Cailliau, Carlos Sagasti, Alvaro J Vis Exp Developmental Biology Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples. Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter (1). We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. MyJove Corporation 2009-02-16 /pmc/articles/PMC2726581/ /pubmed/19229185 http://dx.doi.org/10.3791/1129 Text en Copyright © 2009, Journal of Visualized Experiments http://creativecommons.org/licenses/by/2.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Developmental Biology O'Brien, Georgeann S. Rieger, Sandra Martin, Seanna M. Cavanaugh, Ann M. Portera-Cailliau, Carlos Sagasti, Alvaro Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title | Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title_full | Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title_fullStr | Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title_full_unstemmed | Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title_short | Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
title_sort | two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726581/ https://www.ncbi.nlm.nih.gov/pubmed/19229185 http://dx.doi.org/10.3791/1129 |
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