The overall goal of the following experiment is to observe dividing cells within a developing tissue during C elegance embryogenesis. In order to study genes controlling cell division, this is achieved by creating or obtaining a C elgan strain that stably expresses transgenic markers that label the cells of interest. As a second step selectively knock down a gene of interest by feeding D-S-R-N-A to L four larva for 24 to 48 hours.
The RNAI embryos are collected from graphic hermaphrodites and transferred to an aros pad for live imaging. Next, using wide field or confocal microscopy embryos are imaged to visualize cell division phenotypes. The results will show how neuroblast divide during sea elgin's embryogenesis, and the role of genes required for their division.
This method can help reveal key questions in the field of developmental and cell biology. For example, this method could reveal genes and mechanisms that are involved in regulating mitosis during tissue development, cell cell communication, cell migration, or cell polarity. Use transgenic worms expressing the appropriate markers kept on control or RNAI plates for 24 to 48 hours.
Pick four to six grave adults and transfer them to 20 to 30 microliters of M nine buffer in a depression slide To release the embryos, use a sterile scalpel, cut the worms on either side of the spermatheca or vulva. Next, collect the embryos using a pulled capillary, which is attached to a pipette bulb and is wide enough to pass the embryos. Transfer the embryos to a freshly prepared aros pad using an eyelash glued to a toothpick.
Create groups of five to 10 embryos by pushing the embryos next to each other. Cover the slide with a cover slip and add more M nine buffer if needed, then seal the cover slip with preheated liquid valla. Use an epi fluorescent wide field microscope that includes automated filters.
Objectives, a high res CCD camera and a highly motorized stage. All controlled with software. Although this system does not have them using LEDs and an E-M-C-C-D will limit phototoxicity to the embryo with low magnification.
Manually focus on the embryos to image neuroblast. Increase the magnification and choose embryos undergoing dorsal inter collation or or early the ventral closure on the widefield microscope. Choose the GFP and Texas red filters and determine optimal Z planes on the widefield system.
Using fewer Zacks and fewer time points will reduce phototoxicity also closing the aperture to 30%and if possible, decreasing the light intensity will further reduce phototoxicity. Alternatively, use a live scan swept field or spinning disc confocal microscope with software controlling the scanning lasers objectives, E-M-C-C-D camera, and a highly motorized stage. Now that the acquisition settings have been optimized, image the embryos on the wide field microscope.
It is recommended to film control embryos first before RNAI Embryos. With the swept field system. Set the 4 88 and 5 61 100 milliwatt lasers to 40%power with a slit of 50 and an exposure time of 300 milliseconds.
It is important to note that these settings will be different for other microscopes. Place the slide on the microscope stage, then use a low magnification objective to find the embryos. Then switch to the 60 or 100 x oil immersion objective.
Once focused, collect 20 Z stacks at 0.2 microns every two minutes for a total of 10 minutes. The exposure time will likely require tweaking. Phototoxicity must be considered when live imaging embryos via fluorescent microscopy closing the aperture, choosing fewer Zacks or time points.
Decreasing exposure time can limit phototoxicity if possible. Using a SW field or a spinning disc confocal microscope can help improve this issue. Determine your time, points and Z planes of interest using the acquisition software.
Export desired images as multiple tiffs. Open the desired Z planes for each time point and channel of interest in processing software like Image J and create a stack neuroblast often span only a few planes. Stack the Z planes for each time point and create Z stack projections.
Repeat this for additional channels. Once all of the Zack projections for the desired time points are completed, open the projections to make a time sequence. Do this for each channel separately.
For each channel, make adjustments to brightness and contrast as needed. Rotate the images if necessary. Then select a region of interest and crop the images.
Now make a merged image using the merged channels function. Selecting a different color for each channel. Save the merged image as an RGB file to visualize the images more clearly.
Use the 16 bit TIFF images and select the invert function under edit to create gray scaled images for each channel. To determine the size of an object of interest to microns, draw a line and multiply its length by the size of the pixels, which is determined by the imaging parameters. Now, make a scale bar that represents a proportion of the calculated length in microns.
Ultimately, save the final images as eight bit tiffs. If desired, convert the tiffs to movies using image J epidermal. Morphogenesis of C elegance occurs due to a combination of epidermal cell shape changes, migration and adhesion with help from underlying proliferative neuroblast.
Epidermal cells migrate to the ventral midline and form a single layer of cells to study neuroblast division embryos, stably coex expressing and neuroblast specific marker tagged with GFP and a maternal driven membrane marker tagged with m cherry were imaged during ventral enclosure at increased magnification. Neuroblast were visualized undergoing division neuroblast nuclei are shown in green and the surrounding membranes are shown in red time-lapse images of dividing neuroblast outlined with the mCherry membrane probe in a control embryo were captured using a wide field microscope to better visualize dividing neuroblast in the control embryos. Each channel was kept in gray scale and the images were inverted using swept field confocal microscopy control embryos.
Coex expressing ham one GFP and M cherry pH were imaged at a region with dividing neuroblast. The same embryos treated with RNAI to knock down an ill expression were visualized using wide field microscopy. Unlike control embryos, many neuroblast stalled or regressed during division and became multinucleate using swept field microscopy.
It was also clear that many neuroblast stalled or regressed during division and became multinucleate. This technique can also be used to image migrating cells during tissue formation. Also, if your Zacks are used to permit faster imaging, more information about the subcellular dynamics can be obtained.
Furthermore, with the appropriate equipment, this technique could be used for the activation of photo tuneable probes to perform optogenetics. Also, microscopy techniques such as fret or fret can be applied to study protein, protein interactions or protein dynamics.
