A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

We have developed a mounting method for time-lapse imaging that allows for unrestricted growth of fragile zebrafish embryos and at the same time keeps them in a completely fixed position. This mounting method is fast, easy, and cheap and it works for lab imaging on any inverted microscope. We developed this method for imaging of live zebrafish embryos, but it could be modified for imaging of other small aquatic animal models.

Start by breeding the adult zebrafish. In the afternoon, place the fish into breeding tanks at a male-to-female ratio of one-to-two. Then prepare a stock solution of 1%low melt agarose in embryo media and dissolve it by heating it in a microwave oven.

Aliquot the agarose solution into 1.5 milliliter tubes. Make a stock solution of 4%tricaine in distilled water and store the agarose and tricaine at four degrees Celsius. Tricaine is toxic and has to be weighed and prepared in a fume hood.

After mating, harvest the zebrafish embryos in E3 in a Petri dish and incubate them at 26.5 degrees Celsius for about 28 hours before mounting. Anesthetize the embryos with 0.02%tricaine and dechorionate them under a microscope by gently gripping and pulling the chorion apart to release the embryo. Just before mounting, heat the agarose solution to 65 degrees Celsius and then let it cool to approximately 30 degrees Celsius so the embryo is not harmed by the heat and add tricaine to the agarose solution.

Use 35 millimeter glass bottom dishes with a number zero coverglass bottom. Gently place a dechorionated embryo with one of its lateral sides toward the bottom of the dish with a glass pipette or a micropipette, then carefully remove any remaining E3 with a micropipette. Add the first agarose solution to the small well created by the coverglass attached to the bottom of the dish to cover the embryo making sure that the agarose covers the small well but does not overflow it.

Cover the small well with a coverglass to create a narrow agarose-filled space with the embryo between the two coverglasses. Place a layer of 1%agarose solution on top of the coverglass all over the bottom of the dish which will keep the coverglass in place as it solidifies. Then fill the remaining portion of the dish with E3 containing 0.02%tricaine to keep the system hydrated.

To identify the optimal agarose concentration for layer one, mount the embryos in increasing concentrations of agarose and perform time-lapse imaging to identify the concentration that minimizes embryo distortion and motility, then test a finer range of concentrations based on the results. The most critical step of this protocol is to identify the optimal concentration of agarose for layer one. Test different nano concentrations of agarose such as 0.030%0.032%etc.

to find the optimal concentration. To perform time-lapse imaging of the embryos, use a microscope stage with an incubator set to 28.5 degrees Celsius and image for up to 55 hours. To simultaneously image several embryos, use a stage adapter that holds several dishes and rotate the dishes one by one so that the embryos are horizontally positioned.

Select a low magnification objective, then locate the embryos using the eyepiece and finally align them horizontally by rotating the dishes. Record the embryos’location in the software and create a filename for autosaving the data. Set the pinhole size, scan speed, image size, and zoom.

Then select a higher magnification for capturing images and the fluorescence channels to be imaged. For each channel, adjust the laser power and the detector high voltage making sure to collect the best dynamic range possible while avoiding saturation and limiting photo bleaching. Next, to image the whole embryo with a 10X objective, set the large image capture settings in the software to image several fields of view and stitch them together adjusting the position of the embryo to make sure it fits within this large image area.

Configure the settings for capturing Z-stacks according to the manuscript directions. In order to maintain focus of multiple embryos during long-term time-lapse imaging, use an automated focus and adjust the individual offset levels for each embryo to focus at the reference plane. Then set up time-lapse parameters in the microscope software to image for a duration of 55 hours at one-hour intervals.

At each cycle, capture two embryo datasets sequentially and save data automatically. Use the microscope software to process the images. If more than one embryo was imaged, create independent files for each embryo by splitting the datasets based on the location of the imaging.

Convert the 3D time datasets into 2D time datasets using maximum intensity projections or a similar tool. Finally, create movie files by saving as AVI and selecting the no compression option with 200 millisecond intervals for a playback speed of five frames per second. This layered mounting method makes it possible to image zebrafish embryos while allowing them to grow but at the same restricting their movement.

If the agarose concentration is too high, the embryos become distorted. But if it is too low, they will move out of the field of view during time-lapse microscopy. The optimal agarose concentration was found to be between 0.028 and 0.034%Live transgenic zebrafish expressing RFP in the entire embryo and GFP in the endothelial cells of the vasculature were imaged for 55 hours during which the intersegemental vessel sprouted, subintestinal and head vasculature developed, and caudal vein plexus condensation with trunk extension occurred.

Embryos expressing GFP and motor neurons were imaged to visualize motor neuron development. The motor neuron axons sprout from the ventral neural tube over the somites towards the ventral side of the embryo. The co-development of the dorsal sprouting of motor neuron axons in relation to the position of intersegmental vessels was imaged using a higher magnification.

This made it possible to visualize the finer details of ventral axon sprouting as well as dorsal axon sprouting from the neural tube. As somite numbers increased, the somites also extend in length and width. This process was imaged using transgenic embryos expressing GFP in muscles.

Because the optimal concentration of agarose is very narrow, it is very sensitive to small variations in measurements when preparing the stock solution of agarose and it needs to be redefined for each new batch of agarose solution. It is important to carefully set the imaging parameters including image size and resolution, scan speed, zoom, detector high voltage, and laser power to ensure consistency between experiments and also to reduce the possibility of photo toxicity and photo bleaching during long-term time-lapse imaging. Using this mounting method for transgenic zebrafish embryos followed by extended time-lapse imaging of the whole organism, we can visualize how different tissues develop together.