PUBLICATION
Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
- Authors
- Sagasti, A., O'Brien, G.S., Rieger, S., Martin, S.M., Cavanaugh, A.M., and Portera-Cailliau, C.
- ID
- ZDB-PUB-090302-5
- Date
- 2009
- Source
- Journal of visualized experiments : JoVE (24): (Journal)
- Registered Authors
- O'Brien, Georgeann, Rieger, Sandra, Sagasti, Alvaro
- Keywords
- Developmental Biology, Issue 24, two-photon, confocal, zebrafish, imaging, axotomy, time-lapse
- MeSH Terms
-
- Animals
- Axons/physiology*
- Axotomy/methods*
- Microscopy, Confocal/methods*
- Photons
- Sensory Receptor Cells/physiology*
- Zebrafish/embryology*
- PubMed
- 19229185 Full text @ J. Vis. Exp.
Citation
Sagasti, A., O'Brien, G.S., Rieger, S., Martin, S.M., Cavanaugh, A.M., and Portera-Cailliau, C. (2009) Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. Journal of visualized experiments : JoVE. (24).
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping