To image the behavior of individual cells in vivo plasma DNA containing a cell specific promoter driving expression of a flora four or biosensor is injected into zebrafish embryos. Dynamic changes in effect in distribution are visualized in vivo using a biosensor based on the F effect and binding domain of utrophin. Newly fertilized embryos are lined up along the injection slide and positioned in the corner formed between the top and bottom slides.
DNA encoding fluoro fours or biosensors is injected into the cell. The next day. Embryos are mounted in aros.
On the imaging slide, the embryos are positioned with the labeled cells down and the chamber is sealed with a cover slip. Hi, I'm Erica Anderson from the Laboratory of Mary Hallin and the departments of Zoology and Anatomy at the University of Wisconsin Madison. I'm Nam Tauri, also from the Hallin Lab.
And I'm Matt Clay from the Hallin Lab. Today we will show you a procedure for live imaging of individually labeled cells in zebrafish Embryos. We use this procedure in a laboratory to study cell motility and actin dynamics during axon outgrowth and neuro crest cell migration.
So let's get started. To assemble injection slides first, prepare cyl guard silicone elastomer according to the manufacturer's instructions. Mix 10 parts of the elastomer base with one part curing agent.
Use the cyl guard to bond three standard glass microscope slides together. Arrange two slides side by side at the long edges. Apply ARD to the third slide and place it on top of the other two slides.
Use just enough cigar to seal the slides without excess oozing from between the slides. Any excess can be removed the following day with a razor blade. Let the SIL guard set overnight for imaging slides.
We use stainless steel rectangles cut to the same size as a standard glass microscope Slide with a 1.5 centimeter hole cut in the center. A 22 millimeter squared cover slip is affixed to the steel rectangle with syl guard. Both the injection slides and the imaging slides are reusable and can be cleaned between uses with 70%ethanol, followed by a rinse with water prior to injecting embryos.
Dilute the DNA to a final concentration of 10 to 50 micrograms per milliliter. DNA in 0.2%phenyl red in water. The phenyl red enables visualization during injection.
Next, fill and calibrate the needle. Backfill apol capillary needle with approximately microliters of DNA solution. Wait for the needle to fill and then insert it into the needle holder of the injection apparatus using fine forceps.
Break the tip off the needle such that the tip opening is approximately 10 micrometers in diameter. Measure the diameter of the droplet in mineral oil with an ocular micrometer. To calculate the injection volume, adjust the duration setting of the pico spritzer.
So the droplet diameter is 0.12 micrometers or approximately one nanoliter in volume. A duration of 10 to 30 milliseconds is typical. Collect one cell stage embryos in E three embryo medium and transfer approximately 30 to 60 embryos to the injection slide.
Position the embryos along the edge of the top slide and remove most of the E three so that the embryos are held to the slide by surface tension with a bent dissecting pin. Rotate embryos so the cell is against the wall of the injection slide. Use a micro manipulator to guide the needle through the embryo corion through the yolk and into the single cell of the embryo.
Inject 0.5 to one nanoliters of DNA solution into the cell. Transfer the injected embryos into Petri dishes in E three and place them in an incubator at 28.5 degrees Celsius. If necessary, development can be slowed by incubating embryos at lower temperature on the day after injection.
Prepare the embryos for imaging about an hour before embryos reach the desired age for imaging. Remove the CORs from the embryos with fine forceps. We typically begin imaging embryos at approximately 14 to 17 hours post fertilization.
Next select embryos with labeled cells using a dissecting microscope equipped with fluorescence, we use a Forex objective on an Nikon a Z 100 scope because of its combination of long working distance and high magnification. Place an embryo into a micro fuge tube with approximately 50 microliters of V three. Add trica stock and molten 1%low melting point agros to make a final concentration of 0.02%trica using a wide borg glass pipette.
Immediately transfer the embryo in an agro drop to the cover slip of the imaging slide before the aros hardens position the embryo so the region with labeled cells is facing down against the bottom cover slip. Next, assemble the imaging chamber. Coat one side of a plastic ring with silicone vacuum grease and affix it to the metal imaging.
Slide coat the other side of the ring with grease. Fill the chamber with E three containing 10 millimolar heaps and 0.02%Trica sealed the chamber top by fixing a 22 millimeter squared cover slip to the top greased surface of the ring. The embryo is now ready for four D imaging as will be shown in the next section.
The details of image acquisition will depend on the specifics of your microscope system. Here we demonstrate the time-lapse acquisition process for the Olympus FB 1000 confocal microscope. See previous JO protocol for time-lapse acquisition with the Zeiss LSM five 10 confocal system.
Place the imaging chamber into the slide holder on the microscope stage and focus on the embryo using a 10 x or 20 x objective. Under transmitted light, switch to a 60 x objective numerical aperture of 1.2 or higher, and focus on a labeled cell with epi fluorescence in the FV software. Click on xy repeat to begin laser scanning.
Adjust acquisition settings and image acquisition controls to optimize image to acquire a Z series. Set the upper and lower limits of the Zack along with step size. Engage the depth icon in the image acquisition control window and start acquisition by clicking the XY, Z suite icon to acquire a time lapse series.
Set the time interval and number of time points under the time scan. Heading in the acquisition settings window, click the time icon in the image acquisition control window and start acquisition by clicking the XYZT suite icon. We present here representative movies and images of individual labeled cells.
When individual neurons are labeled, their axons are not obscured by other labeled cells and thus the OD extensions on axons and growth cones are clearly visible. This movie shows a sensory neuron labeled with membrane targeted tag RFP. It extends two axons in opposite directions that grow out of the field of view.
A third axon extends orthogonally from the cell body and forms branches. Similarly, labeling of individual neural crest cells allows imaging of fine cellular processes associated with cell motility. Here two neural crest cells are labeled with membrane targeted GFP.
They exhibit extensive protrusive activity while migrating in an anterior direction. It is often useful to label individual cells within a transgenic line to visualize interactions between neighboring cells within a population. This image shows an embryo with stable expression of GFP in all sensory neurons that was injected with DNA encoding tag RFP to label one neuron red in the background of green neurons injection of DNA encoding.
The actin biosensor probe shows stronger effect in signal in growth cones and dynamic protrusions along newly formed axons compared to the cell body. At a later stage signal remains strong in growth cones while mature axons have significantly weaker signal. We've just shown you how to perform live imaging of individually labeled neurons and neural crest cells in zebrafish embryos.
When doing this procedure, it's important to choose embryos in which the DNA is not expressed at levels high enough to cause toxicity or cell death. So that's it. Thanks for watching and good luck with your experiments.
