Developmental Biology: Zebrafish Applications

Explore whether widefield or confocal imaging best suits your microscopy needs

The Cytation C10 confocal imaging reader makes capturing both widefield and confocal fluorescence images simple and automated. Confocal imaging excels at eliminating light originating outside of the focal plane, but it is important to recognize that not all samples benefit from this reduction for analysis. In the example shown in Figure 1A, this evaluation required capturing a large section of the vascular structure of the zebrafish tail into a single image. This transgenic zebrafish line expresses fluorescence only in the region of interest (vasculature), with essentially no background signal. Here, the confocal modality is not advantageous over widefield for this purpose: a single image using lower magnification and widefield optics rapidly produces the high-quality images necessary for analysis Depending on your sample characteristics, widefield imaging could potentially result in shorter image acquisition times.

On the other hand, the sample in Figure 1B clearly benefits from imaging with the confocal modality. Here the experiment calls for identification of a single motor neuron deep in the zebrafish tail, and the immunostaining protocol used generates staining in other neuron types above and below the focal plane of the cell of interest. This out of focus light in the widefield images interferes with the segmentation of the motor neuron, whereas the confocal modality achieves proper identification of the motor neurons.

Figure 1. Widefield and confocal fluorescence microscopy applications for zebrafish phenotypic analysis. (A) Widefield fluorescence quantifies total vascular area as shown in this transgenic zebrafish expressing red fluorescence throughout the vasculature. Here widefield is preferred due to the low background fluorescence and large field-of-view (low magnification) desired to capture a large portion of the vascular structure. (B) Confocal fluorescence is preferred to appropriately segment individual neurons (red staining) in this immunofluorescence zebrafish application. For this sample, widefield images include too much out-of-focus background fluorescence to properly segment motor neurons.

Automate routine fluorescence sorting tasks that don't require imaging

The small size of zebrafish embryos makes them highly amenable to higher throughput formats, such as 96-well microplates. This enables automation of routine tasks like sorting fluorescent embryos from non-fluorescent siblings. Depending on the onset time of fluorescence, embryos can be sorted in their chorions without anesthesia, or immobilized with anesthetics at later stages (post-hatching) for sorting. Round-bottom microplates can be used to effortlessly and automatically position the embryo in the center of each well. Fluorescence detection using plate-reader monochromators can identify fluorescent embryos, such as the transgenic GFP-expressing embryos pictured in Figure 2. Here, 1-day old embryos were arrayed into a round bottom 96-well microplate for sorting by using reader-based fluorescence on the Cytation C10.

Figure 2. Automate detection and sorting of live fluorescent zebrafish embryos.

Images show arrayed 1-day old zebrafish embryos positioned in the center of the well in a 96-well microplate. Fluorescence detection of all 96 embryos takes approximately one minute, and a cutoff threshold is then applied to identify GFP positive fish from non-expressing siblings.

Application Notes

• Using Acridine Orange to Measure Cell Death In Ethanol Treated Zebrafish Embryos
• Analyzing Wound Healing in Zebrafish Embryos
• Automated Image Screening of Zebrafish Embryos Exposed to Developmental Toxins
• Quantitative Microscopy of Angiogenesis in Zebrafish Embryos