This protocol demonstrates how to image dynamic events in early CL egan's embryos with Norky DIC optics or fluorescence by making use of widely available microscopes and open source software. The protocol begins with describing how to dissect CL gans embryos and mount them for imaging. Then proper setup of the DIC nor masky optics is reviewed to ensure optimal images at the highest detail.
Calibration of wide field epi fluorescent and illumination is also reviewed for collecting good fluorescent image data teamed with open source software. Good microscopy can visualize cellular dynamics in a developing embryo. Visual illustrations of this method is critical as setting up D iic Optics properly is hard since the terminology can be confusing and the proper technique is hard to describe in written form.
In addition, researchers should be careful to minimize light exposure when performing fluorescent imaging. In order to limit phototoxicity and photobleaching To prepare embryos for imaging, first, make two mounting solutions. One is melted Vaseline and the other is two to 3%aros in buffer.
Denser aros can be made if the structures to be imaged are very fragile, being careful to avoid boiling. Both stock solutions are melted in the microwave and then stored as four to five milliliter aliquots. On the day of imaging, melt an aliquot of Vaseline solution in a 65 to 70 degrees Celsius heat block and melt one aliquot of arose solution in the microwave.
Again, avoiding boiling. Then to keep these solutions molten, store them in a heat block. Next, cover the top and bottom of two slides with a strip of lab tape and position a clean slide between the two taped slides.
Now pipee a few drops of the molten aros onto the clean slide and cover it with another clean slide. The Aros spreads into a thin square pad with the worm pad prepared. Use a dissecting scope and a platinum wire pick to transfer two adult worms to a nine to 12 microliter drop of buffer on an 18 by 18 millimeter cover slip.
Then cut. Open the worms with a needle or scalpel to release the embryos with the agarro side down. Position the worm pad onto the 18 by 18 millimeter cover slip and razor away any overhanging agar.
Finally, to seal the cover slip, apply the molten Vaseline solution along its perimeter with a paint brush or toothpick. The cells are now ready for imaging. When putting the slide on the microscope, make certain the two polarizers are aligned perpendicularly to each other.
Ensure that both wallers and prisms are out of the light path. If the field is not dark, rotate the polarizer below the condenser until it is now. Find the embryos using the 10 times objective.
Look near the worm bodies for groups of young embryos in order to get multiple embryos within the imaging field. Avoid embryos inside the worm for the best images. With the best embryos located, switch to a higher power objective.
Now switch the filter cube to DIC. Next, insert the upper objective walstone prism into the light path and rotate the tart on the condenser to select the lower walstone prism that matches the objective. With prisms in place, adjust the slider below this turret to match the numerical aperture of the lens.
To get the optimal Kohler illumination, focus the condenser until the edge of the condenser diaphragm appears Sharpest. Sharpest is when the border between light black is most crisp or in focus. To get optimal contrast, adjust the apple wall and prism by rotating the knob on the slider.
Now the microscope is nearly ready to use. It is time to control additional imaging parameters on the computer with micromanager and open source software. Begin by setting the camera gain to zero.
Then switch to saving files as eight bit, not 16 bit tiffs. TIFF files can later be analyzed by open source software like Image J.Next, adjust the light level to where the brightest pixels are just below saturation levels. If everything has been done correctly, the image will look 3D with light appearing to shine in at an angle.
Now that the image looks brilliant, select the embryos you want to image and draw a region of interest. If the microscope has a focus motor, use it to collect multiple focal planes at each time. Point open the multidimensional acquisition window and set the Z stack top and bottom plane by adjusting the focus knob to find the first and last focal plane with some yoke granules in focus.
Set the step size. This will define the number of focal planes collected. Now, set a time interval that is long enough to acquire all of the focal planes.
The maximum time points can be set very high so that the software takes images until it is manually stopped. Set the software to display only the last acquired image so that the process can be monitored. Now give the images a file name and begin acquiring images.
Image analysis will be briefly reviewed at the end of the next section. Constructing a GFP movie is much like making four D nor Musky DIC, but a configuration file that includes software control of fluorescent shatter is used. Instead with the embryos located, make sure that the upper walstone prism is not in the light path and switch the filter cube to F-I-T-C-G-F-P.
If this was not automatically set On startup using the software, make sure the camera gain is set to 255. An image file is set to 16 bit tiff format. Now turn off the transmitted light source and click live imaging.
Adjust the intensity of the UV light source and exposure time to achieve good signal to noise ratio for the fluorescence signal, however, keep the light exposure at a minimum to preserve the cells. Next, set up the time interval. The number of time points and the number of focal planes now proceed with image acquisition as previously described.
To analyze the movies, use open source software. Image J with the micromanager plugin installed Very large movies can also be imported into Image J using the loci plugin. This is a wild type embryo captured with nor masky, DIC optics.
Posterior is to the bottom right and ventral is to the lower left. The movie shows a sequence of single focal plane images collected every 15 seconds. Playback is set at 14 frames per second.
This embryo expresses fluorescence during the first embryonic division here posterior is to the upper right. This movie shows the changes in a single focal plane every 10 seconds. After watching this video, you should have a good understanding of how to image live embryos using both DIC Naski optics and epi fluorescence to study dynamic events.
We hope you have a better understanding of how to set up DIC Nomar optics to get optimal images and realize that you can get high quality fluorescent data using epi fluorescence illumination, and are not always dependent on more advanced microscopes. You can also apply these imaging techniques to examine embryos lacking specific gene function to further investigate the genetic requirements for your favorite events during cell division or development.
