The overall goal of this photoconversion technique is to distinguish single cells in a single animal from other fluorescent cell populations. This method can help answer key developmental questions in the field, and allow us to visualize single-cell migration over time. The main advantage of this technique is that it is minimally invasive and can be used to label single cells or larger populations.
Additionally, the user can select the specific region of interest and photo convert the desired target. After mating male and female transgenic zebrafish expressing a photoconvertible protein and collecting the eggs according to the text protocol, use a dissecting microscope with a 488 nanometer light source and GFP filter sets to screen 24 hpf embryos. Using a needle or tweezers, manually dechorionate the embryos.
Prepare in microwave, five milliliters of 0.8%low melting point agarose solution. Once the agarose is cooled to the touch, place three to four anesthetized transgenic sox10 eos positive 48 hpf fish in the center of a ten millimeter glass cover slip bottom Petri dish. Add approximately one millimeter, or enough agarose to cover the surface of the cover slip.
Then, use a probe needle to arrange the fish on their sides, adjusting them as needed, while the agarose solidifies, which will take about two minutes. After the agarose has solidified for two minutes, slowly add embryo medium containing 0.02%aminobenzoic acid ester tricaine to the dish, until the bottom surface of the agar and dish is submerged. To carry out confocal microscopy, open confocal software and select the capture and focus windows.
To carry out pre-conversion imaging, under the capture window and capture setting dropdown tab, select the imaging parameters. Here are the lab specific stack setting is called, fish imaging. Place the specimen on the confocal microscope and use the coarse and fine adjustment knobs to bring it in to focus.
Then open the focus window and locate the desired region of interest, such as, the dorsal root ganglion. Under the filter set menu, select the c488 laser, set the exposure to 300 milliseconds, the laser power to five, and the intensify to 75. Next, under the capture type section, check the 3D box.
In the 3D capture section, select use current position, and check range around current. In the same section set the range to 35, the number of planes to 36, and the step size to one. Select the current location, then click start at the bottom of the capture window to acquire an image.
Before the photoconversion process, be sure to double and triple check the laser parameters. Sometimes it's necessary to readjust the conversion settings, when the conversion window is open. In the capture window, under the capture window setting dropdown tab, select the lab specific conversion imaging setting.
Here the lab specific setting is called, fish ablate full chip. Under the filter menu set, select the c488 and c541 lasers. Set the exposures to 300 milliseconds, the laser power to five and the intensify to 75, which will produce enough fluorescent signal without causing photobleaching or toxicity.
In the focus window, click on the photomanipulation tab, and change the laserstack power to two and then click go. Change the raster block size to one and click set. Then change the double-click size to four and the laser line to v405.
Next, in the capture window open the advanced capture settings. Select the photomanipulation tab and change the double-click repetitions to two, then click okay. Select the xy tab in the focus window.
In the photomanipulation tab, double check the laser parameters. Then, set laser settings to photoconvert the cell of interest, without photoconversion of the surrounding cells. Under capture type, check the timelapse box, then click start.
Once the live timelapse window opens, select the circle tool on the top toolbar. Draw a circle in the centermost region of the cell, then right-click the drawn circle, select FRAP Region, and wait three seconds. The selected area should become dimmer, when it does click stop capture.
If imaging more than one animal, go back to the xy tab in the focus menu and select position two. Then, repeat the photoconversion. To convert a population of cells, follow the protocol and laser perameters through opening the timelapse window.
Now, instead of drawing a circle to FRAP the region of interest, use the line tool to draw a line on the region of interest. Then, FRAP the region using the same parameters just demonstrated. Once all points are photoconverted, under the capture setting dropdown tab in the capture window, select the lab specific standard stack setting described earlier in this video.
Select the c488 laser and set the exposure to 300 milliseconds, the laser power to five, and the intensify to 75. Select the c541 laser and set the exposure to 500 milliseconds, the laser power to ten and the intensify to 75. Under the capture type section check the 3D box.
In the 3D capture section, select use current position, and check range around current. In the same section, set the range to 35, the number of planes to 36, and the step size to one. The range number may increase or decrease, to accommodate the desired imaging depth.
If there are multiply points in the xy focus tab, in the capture window, select the multipoint list option. If not, select current location. Finally, at the bottom of the capture window, click start to acquire an image.
These panels show a single prephoto conversion cell, within a ganglion from a transgenic zebrafish, expressing the photoconvertable eos protein, under control of the sox10 regulatory sequence. Following photoconversion, eos was distinctly present within the region of interest, while no neighboring cells were labeled. To demonstrate the utility of this photoconversion technique, a population of cells in the ventral side of the spinal cord was exposed to UV light, using a line.
Afterwards, images were taken showing the photoconverted eos protein in the region of the line. Non photoconverted eos protein was visible in all other areas. Several hours after the photoconversion, identical images were taken that showed photoconverted eos positive cells scattered throughout the spinal cord region, in both dorsal and ventral locations.
This finding is consistent with the hypothesis that, ventral spinal cells relocated to dorsal locations. Once mastered, this technique can be done in 30 to 45 minutes, if performed properly. While attempting this procedure, it's important to remember to take a postconversion image, to confirm that your region of interest did experience photoconversion.
Following this procedure, other methods like, timelapse imaging can be performed in order to observe cellular migration and dynamics over time. After watching this video, you should have a good understanding of how to distinguish single cells within a fluorescent cell population. Don't forget that working with ultraviolet light can be extremely hazardous, and necessary precautions should always be taken while performing this procedure.
For example, do not ever stare directly into the light.
