Zebrafish are an important model organism for biomedical research. Structural, functional, and genetic similarities with humans make zebrafish a useful model for phenotypic screening in drug discovery and toxicology testing. They are small, develop at a rapid pace, and as vertebrates they share many of the same organs and cell types as mammals.1 Zebrafish are a great model organism to use for imaging, in large part due to their optical clarity, which makes imaging multiple processes in disease and development possible. In this article we will discuss considerations for embryo preparation, vessel and mounting choices, and imaging in order to obtain quality data.
During embryo preparation remember to be gentle with the embryos in order not to damage them and destroy your sample. Also, keep them out of light as much as possible, as light will cause the yolk sac to darken. Tricaine is a common method of anesthetizing embryos. A small amount of tricaine will keep embryos immobile for hours, and is especially effective if combined with a mounting method such as low melt agarose that will physically restrain the embryos. Be careful not to add too much tricaine however, or there is a risk of killing the embryos. Usually adding a couple drops of a 0.4% stock tricaine to a dish is sufficient or a 0.016% working solution can also be prepared.
Figure 1: 1 day post fertilization (dpf) zebrafish embryo stained with myl7 (gfp). Shown is a focus stack of the gfp and brightfield channels.
The choice of vessel used to image the zebrafish depends a great deal on the type of assay being run and how the embryos need to be orientated. High-throughput assays are typically run on 96–384 well plates. Lower throughput imaging can be performed on slides or larger dishes where it is easier to orientate embryos in order to capture the desired area.
Round bottom plates
A benefit of using round bottom plates is that the embryos tend to settle in the center of the well, making it easier to find the embryo for imaging. A downside is that embryos will not lie perfectly on their sides as easily with the sloped bottom.
Flat bottom plates
A benefit of using a flat bottom plate is that the embryo will more readily orientate to a lateral position. A downside is that the embryo will tend to collect in the edges of the well, making it necessary to image the whole well or set up beacons in a different location in each well.
Slides
There are multiple methods for preparing microscope slides for zebrafish imaging. The main idea is that there needs to be a barrier between the slide and the coverslip to prevent the embryo from being flattened during mounting. One method is to create an imaging chamber with layers of electrical tape. To accomplish this, layer about three pieces of electrical tape on top of each other, and then use a razor blade to cut a square in the middle of the tape, creating a well for the zebrafish to lay in. Additional or fewer layers of electrical tape may be employed as necessary. The electrical tape square is then adhered onto a slide, and the embryos can be mounted in this reservoir. This will keep from damaging the embryo when placing a coverslip onto the slide.
Proper positioning of embryos is critical for successful imaging and there are various media that one can use to immobilize the zebrafish into the proper position for imaging.
Methylcellulose
Live embryos can be mounted in 2–3% methylcellulose, which is rigid enough to keep the embryo in place long enough for imaging, but also allows for manipulation.
Low melt agarose
Low melt agarose is a good choice for long term imaging as it keeps the embryo firmly in place. However, be careful of the temperature when mounting live embryos as agarose that is too hot can kill the embryos. After preparation of agarose, allow to cool to an appropriate temperature before mounting the embryos.
Glycerol
Glycerol can also be used to mount fixed embryos. Similar to methylcellulose, glycerol is viscous enough to keep the embryo in place, but also allows for the embryos to be manipulated into multiple positions if necessary. Typically, 80% glycerol is a good choice as 100% glycerol can be too viscous to work with.
Imaging a whole zebrafish embryo can be challenging as the entire embryo will not be in the same focal plane. The thickness of an embryo can also make automatic focusing difficult. One way to overcome this is by taking image stacks. The stacks can then be processed, such as into a focus stack to get a nice picture of a brightfield embryo, or maximum projection stacks which can be useful for analysis of fluorescent images.
In conclusion, zebrafish are a great model organism for imaging, due to their size and optical clarity, but processing and imaging them properly sometimes require trial and error. These tips will help you chose the best vessel and mounting method for your imaging needs.
1. Rubinstein, A. L. Zebrafish: from disease modeling to drug discovery. Curr Opin Drug Discov Devel 6, 218–23 (2003).