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

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. 1. Prepare 1% low melt agarose solution for embedding. Dissolve agarose in DI water by heating in a microwave, aliquot into small tubes and store in a heating block at 42 degrees Celsius. 2. Select embryos for imaging and remove their chorions by gently pulling the chorion apart with forceps. Embryos can be placed into a 5% PTU Ringers solution at 22-24 hours post fertilization (hpf) to inhibit the formation of pigment. This improves the clarity of imaging and is essential for performing two-photon axotomies of sensory axons. If the natural pigment is allowed to form, autofluorescence obscures axons on the two-photon microscope. Ringers solution for zebrafish contains 116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, and 5mM HEPES, pH 7.2. 3. Anesthetize embryos by adding~0.02% tricaine to the PTU Ringers. Make sure animals are not responsive to touch before proceeding. 4. Prepare the long-term imaging chamber by applying vacuum grease to one side of a glass or Teflon ring and fixing it onto a glass cover slip. 5. Transfer one embryo into the 42 degree agarose solution with a glass pipet, taking care not to transfer much of the PTU Ringers, then transfer the embryo with one drop of agarose onto a prepared cover slip. 6. Position the embryo as desired as the aga...

Graded levels of Pax2a and Pax8 regulate cell differentiation during sensory placode formation

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Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning

“…Embryos were dechorionated, anaesthetized in 0.01% tricaine and mounted in a sealed agarose chamber (O'Brien et al, 2009). A custom-built two-photon microscope equipped with a femtosecond Ti:Sapphire laser (Chameleon Ultra II, Coherent) was used to image and axotomize GFP-labeled trigeminal neurons, and images were collected using ScanImage software (Pologruto et al, 2003).…”

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