The overall goal of the PSmOrange Photo Conversion Technology is to track cells in the living animal during embryonic development and disease. This method can answer key questions in developmental biology such as uncovering the origin of cells which develop into tissues and organs. The main advantage of this technique is that it can be applied in GFP transgenic backgrounds.
This method can also be applied in different fields of investigation such as regeneration, tumor progression and invasion. After generating and embedding PSmOrange and GFP expressing embryos according to the text protocol, place the embryo under the confocal microscope and use the 20x air objective and 488 and 561 nanometer lasers to scan the specimen to identify an area to photoconvert. To detect the PSmOrange protein before photoconversion, use the 561 nanometer laser at 0.74 milliwatts of power measured at the focus plane above the objective and adjust the laser power as necessary.
Adjust the zoom factors to highlight the area of interest and reduce image acquisition time and photo toxicity. Using the 488 and 561 nanometer lasers in sequential mode and a Z step between 1.0 and 2.0 micrometers, acquire a Z stack covering the structures of interest. Adjust the line average and scan frequency to optimize the image quality.
Then scan the sample and select the region of interest, or ROI tool, to highlight the area to photoconvert before fixing the ROI as the stimulation area. Select the photoactivation bleaching module, check the box to activate the 488 nanometer laser, then set the 488 nanometer laser to 80%and the scan speed to 0.5 seconds per frame. Next, open the photoconversion utility and click on Add'to specify the photoconversion protocol.
In the acquisition stimulation menu, set Phase One to acquisition and in the loops menu, indicate the number of images to be acquired before photoconversion. Set Phase Two to stimulation and enter the number of stimulation events to be performed. Adjust Phase Three as described for Phase One.
Optionally, insert a Waiting Phase after Phase Two by entering the time to wait before the last round of image acquisition. Once the parameters are set, apply the stimulation setting and run the photoconversion. Miscroscopic settings are crucial for PSmOrange photoconversion.
To ensure a successful photoconversion, the embryo is imaged directly after the experiment to detect the photoconverted PSmOrange protein. If the conversion was not successful, the procedure is repeated. Using the 488, 561 and 640 nanometer lasers, and setting the 640 nanometer laser to high power, up to 4.5 milliwatts, acquire a final z stack with the settings just described.
To dismount the embryo, remove the chamber from the microscope and use forceps to remove the embryo from the agarose. Transfer the embryo into a new sterilized plastic petri dish, or sterilized 6-well plate, containing 0.2 millimolar PTU in E3.Incubate the embryo at 28 degrees Celsius until the desired stage of development. To analyze the fate of photoconverted PSmOrange protein expressing cells, re-embed the embryo according to the text protocol.
Under the confocal miscroscope, scan the specimen to identify photoconverted cells in the GFP transgenic structure of interest. Use the 488, 561, and 640 nanometer lasers to acquire a z stack with a fixed stack of 1 to 2 micrometers in sequential mode covering the structures. Using Image J Fiji software, identify cells which co-express GFP and the photoconverted protein.
Duplicate the original stack, use the Gaussian Blur plugin and the image calculator to subtract the smooth stacks from the original. Apply specific thresholds to the green and far red channels in order to highlight the photoconverted cells. Then use the Analyze Particles tool to detect the threshold areas in the green channel with a suitable setting.
Repeat this step for the far red channel. Open the image calculator plugin to detect the co-localizing cells. Display in yellow the overlapping ROIs in the green or the far red channel using the Analyze Particles tool.
Using NIS-Elements software, carry out 3D date evaluation and display the stack in 3D using the Show Volume View option. Then use the graphic interface to select the green, red, and far red channels, adjust the brightness and the contrast and crop the 3D stack to highlight the photoconverted area. Shown here is an example of the PSmOrange photocoversion system in zebra fish transgenic FOXD3 floating head GFP embryos.
At 26 hours post-fertilization, two cell clusters in the anterior part of the pineal were photoconverted and immediately imaged. The protein was detected with a 640 nanometer far red laser but not with a 561 nanometer laser. In these cells, GFP expression was initially reduced due to photobleaching.
At 52 HPF, GFP and H2B PSmOrange were detected in the photoconverted H2B PSmOrange protein expressing cells. A few of these cells formed the parapineal on the left side of the brain. This result is consistent with previous reports on the parapineal cell origin in the anterior pineal and shows the applicability of the PSmOrange technique in the living GFP transgenic vertebrate embryo.
Following this procedure, the cell tracking analysis can be performed in genetically manipululated embryos in order to answer additional questions regarding the molecular hierarchies underlying the cell's development.
