The overall goal of the following experiment is to photo activate a given light responsive agent, such as chemically caged fluorescent dye in a live zebrafish embryo at a single cell resolution. This is achieved by injecting a light responsive agent into one cell stage embryos expressing a live genetic landmark to locate and precisely target any cells of interest. As a second step embryos are grown and mounted in a low melting point aeros, which immobilizes the live embryo.
Next two photon microscopy is used in order to localize a visible fluorescence transgenic landmark. The light responsive agent is then activated in a desired focal plane and the extent of photo activation is monitored. The fate of the resulting labeled cells can then be traced at later embryonic stages.
This procedure can be utilized for the activation of a variety of compounds at a single cell resolution in live specimens. The the main advantage of this technique of existing methods, such as confocal microscopy or flash lamp, is that it allows photo activation of relatively deep tissues at axial level of a few microns with relatively low Phototoxicity. But the method presented here is used for seline edge tracing and it can be applied for photo activation of a variety of components, and that's allowing targeted control over developmental and physiological processes On the evening before the injection prepare mating tanks separate male and female zebrafish of a transgenic line expressing a visible fluorescent landmark.
In this experiment, the transgenic line expresses GFP under the control of neurogenic N one promoter thaw an aliquot of 5%dextran conjugated caged fluorescein in 0.2 molar potassium chloride on ice dilute the caged fluorescein in 0.2 molar potassium chloride to a final concentration of 1%Keep the fluorescein on ice and protect it from light. Allow the zebra fish crosses to mate collect the fertilized eggs as soon as they're laid and rinse them as described in the text. Use a plastic pipette to transfer one cell stage embryos to a 1%aros injection plate that has been overlaid with fresh E three medium under a dissecting microscope.
Use the pipette to orient the embryos in the injection troughs. Break the tip of a capillary injection needle that has been pulled to a long taper. Use a micro loader tip to backfill the needle with approximately one microliter of 1%caged fluorescein.
Place the loaded needle into a microm manipulator attached to a pneumatic micro injector. Inject each embryo with two to three nanoliters of caged fluorescein directly into the cell's cytoplasm. Inject at least 50 embryos per experiment.
Transfer injected embryos to a Petri dish containing fresh E three medium incubate in the dark at 28.5 degrees Celsius to the desired developmental stage to inhibit pigmentation at 0.1%Phenyl threa to embryos at 24 hours to prepare for embryo mounting. Coat 15 to 2060 millimeter Petri dishes with a thin layer of 1%Aros dissolved in fresh E two medium when the aros has solidified. Fill the dish with E two medium under a dissecting microscope.
Use sharp forceps to coate injected embryos on coated plates. Avoid exposing the embryos to air. Keep an aliquot of 2%low melting point aros in water.
In a heat block at 72 degrees Celsius. Use a fire polished past pipette to transfer one coated embryo in approximately 150 microliters of medium to a 60 millimeter petri dish. Place a similar volume of 2%aros beside the embryo and quickly mix the two droplets together to obtain a homogeneous 1%Agros droplet.
Orient the embryo with its dorsal side facing the objective lens. Allow the aros to solidify submerge the embedded embryo in E two medium. Place the freshly mounted embryo on a plate holder under a two photon microscope equipped with a broadband turntable laser.
Before beginning the photo activation process, acquire an image series from the most ventral to dorsal edge of the expressing tissue at 860 nanometers and space. The images six microns apart on the Z plane. Select the target Z plane for photo activation and bring it into focus using GFP as a landmark.
Locate the region of interest. Take a single image at 860 nanometers and keep it open in a separate window for proper alignment. It is crucial that the laser at 720 nanometers be in a mode locked state before opening the edit bleach menu.
Use the image taken at 860 nanometers to define the region of interest or ROI for photo activation. In the edit bleach window, adjust the relative laser power number of iterations and scan speed. For photo activation, press bleach when the laser has stopped, switch back to 860 nanometers and acquire a single plane image to evaluate the intensity of the resulting photo activated material.
Collect an image stack to evaluate the zsan or thickness of the activated domain. In this way, it is possible to empirically determine the laser intensity and duration of exposure. For optimal photo activation of each clone or cross photo, activate all of the remaining embryos for the experiment.
Using these conditions, raise the mounted photo activated embryos in the dark at 28.5 degrees Celsius in E two medium containing 0.1%Phenyl thio urea until they have reached the desired developmental stage. Under a dissecting microscope, use a pointed scalpel to make a V-shaped notch in the aros near the mounted embryo with the bottom of the notch pointing toward the embryo's head. Place a set of closed forceps at the point of the notch.
Gently open the forceps separating the aros along the length of the embryo and releasing the embryo into the medium. After releasing each embryo, use a fire polished pasture pipette to collect it into a micro centrifuge tube. In a chemical fume hood, fix the embryos with 4%formaldehyde for three hours.
At room temperature, proceed with dehydration, rehydration staining with alkaline phosphatase, conjugated, anti fluorescein, and all associated washes as described in the accompanying text. Add 500 microliters of filtered fast red solution to the embryos. Incubate in the dark at room temperature replenish with new fast red staining solution Every hour until the desired signal to background ratio is reached.
Stop the staining reaction with three washes in PBST of five minutes each. In the fume hood, fix the stain in 4%formaldehyde for 20 minutes. Wash twice in PBST for five minutes each.
Wash clear embryos through a series of 25%50%and 75%glycerol gradients in PBS until each embryo is settled at the bottom of the tube. Store the embryos at four degrees Celsius until ready for visualization on the microscope. To mount the embryos for visualization, use a one milliliter syringe to apply four 20 microliter spots of clear silicone grease on a microscope slide at the corners of a 15 millimeter square shape.
Place an embryo in approximately 200 microliters of 75%glycerol between the drops of grease. Place a cover slip over the embryo. Gently push the cover slip straight down until the glycerol solution fills.
The space between the drops of grease mounted embryos can be stored for a few days in four degrees Celsius before visualizing with confocal microscopy. This image shows a live zebrafish embryo expressing GFP under the control of neurogen. In one promoter that was injected with caged fluorescein tracer dye at the one cell stage using the preceding method at the three to five so mite stage.
A discrete area of the four brainin primordial was photo activated by two photon laser and the uncaged fluorescein tracer dye could be detected. Subsequent to the photo activation procedure, the embryo was incubated at 28.5 degrees Celsius, and brain cells containing the uncaged fluorescein were traced by anti fluorescein immunostaining in red at 24 hours post fertilization. Note the small number of cells labeled Once mastered the photo activation procedure can be performed in just a few hours While attempting this procedure, it's important to remember to control the optimal parameters, allowing precise photo activation at a single cell resolution.
