The overall goal of this procedure is to track photo switched cells in a living zebra fish embryo. This is accomplished by first embedding an embryo, expressing a tissue specific photo, convertible fluorescent protein in low melting temperature aros. Next, the protein within the region of interest is completely photo converted.
Then the embryo is carefully removed from aros and maintained in egg water at 28.5 degrees Celsius until later stages. Finally, the photo converted embryo is again embedded in low melting temperature aros. Ultimately, results can be obtained that show that photo converted cells can precisely be detected at later stages due to the stable presence of the photo converted protein through confocal microscopy.
So we think that the method that we will present today is going to be quite useful in the context of cell and developmental biology. So we think that using this methodology in the context of cell migration, tissue dynamics, and other cell behaviors will be quite useful. Demonstrating the procedure will be Veronica Lombardo, a postdoc from my lab After crossing transgenic zebrafish, expressing a photo convertible fluorescent protein.
Choose the brightest embryos which are homozygous and grow them at 28.5 degrees Celsius when they've reached the desired stage. Use forceps to dec coate them in a glass container with egg water to anesthetize the embryos. Use a glass pipette or a cut pipette tip to transfer them one at a time into trica.
Transfer an anesthetized embryo onto the cover of a culture dish and maintain the embryo within the smallest possible volume of medium. Transfer the embryo into aros by pipetting it with 1%low melting temperature agros with trica at 30 degrees Celsius, and place it into a cover glass bottomed culture dish. Use a smooth plastic tip to orient the embryo when the agros has polymerized.
Cover it with Trica solution on an inverted confocal microscope equipped with 405 488 and 561 nanometer laser sources and a 20 x objective. Use the 488 and 561 nanometer lasers to generate a Z stack of the entire structure of the specimen, which includes the region of interest. Select the time series tool and set for two cycles with no interval, one for pre and one for post photo conversion.
Select the region's tool and define one or more regions of interest for photo conversion. Next, select the bleaching tool and set for start bleaching after scan. One of two used here is a 30 milliwatt 405 nanometer diode laser.
Carefully optimized for the minimal amount of laser light necessary for complete photo conversion, which is achieved by varying the intensity of the laser light, the scan iterations of the region of interest and the scan speed. Scan the sample with the 488 nanometer and 561 nanometer lasers to visualize any remaining green and the photo converted red fluorescence of the photo convertible fluorescent protein. After photo conversion, discard the solution and using a needle or smooth plastic tip carefully remove the embryo from the agros.
Maintain the embryo in egg water in the dark at 28.5 degrees Celsius until the desired developmental stage. Embed the embryo a second time, then generate a Zack of the specimen, including the region of interest, again using the 488 and 561 nanometer lasers. Because this protocol does not affect normal development, it is possible to observe the photo converted embryo at later stages.
Here is an example of a photo conversion assay. Endothelial cells were tracked during zebrafish development from 48 to 72 hours post fertilization using a transgenic reporter line expressing kick G within endothelial nuclei as shown here before photo conversion kick G was expressed within endothelial tissue and was only detectable as green fluorescence using a 488 nanometer argon laser control scans with a 561 nanometer laser for red fluorescence. Did not detect any photo converted kick GR prior to the photo conversion.
Subsequently, the region of interest was completely photo converted using a 405 nanometer diode laser at 10%intensity, 20 iterations and the head vessels were scanned again using the 488 nanometer and 561 nanometer lasers. In this panel, green kick G had completely switched to red within the region of interest to track the photo converted endothelial cells at later stages. The embryo was observed at 24 hours post photo conversion, which is approximately 72 hours post fertilization within endothelial tissues arising from the photo converted region of interest.
Both non and photo converted kick G proteins were observed due to the stability of the red fluorescent photo converted kick g, and synthesis of new green non-converted kick G After its development. This technique paced the way for researchers in the field of developmental biology to explore cell biology and tissue remodeling in several fish.
