Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Georgeann S. O'Brien, Sandra Rieger, Seanna M. Martin, Ann Cavanaugh, Carlos Portera-Cailliau, Alvaro Sagasti

Research output: Contribution to journalArticle

40 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere1129
JournalJournal of Visualized Experiments
Issue number24
DOIs
StatePublished - Jan 1 2009
Externally publishedYes

Fingerprint

Time-Lapse Imaging
Axotomy
Zebrafish
Photons
Microscopes
Embryonic Structures
Imaging techniques
Axons
Sensory Receptor Cells
Neurons
Phenylthiourea
Ultrashort pulses
Sepharose
Regeneration
Lasers
Recovery
Wounds and Injuries

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A., Portera-Cailliau, C., & Sagasti, A. (2009). Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. Journal of Visualized Experiments, (24), [e1129]. https://doi.org/10.3791/1129

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. / O'Brien, Georgeann S.; Rieger, Sandra; Martin, Seanna M.; Cavanaugh, Ann; Portera-Cailliau, Carlos; Sagasti, Alvaro.

In: Journal of Visualized Experiments, No. 24, e1129, 01.01.2009.

Research output: Contribution to journalArticle

O'Brien, Georgeann S. ; Rieger, Sandra ; Martin, Seanna M. ; Cavanaugh, Ann ; Portera-Cailliau, Carlos ; Sagasti, Alvaro. / Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. In: Journal of Visualized Experiments. 2009 ; No. 24.
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